Double-acylated glp-1 derivatives

ABSTRACT

The invention relates to a derivative of a GLP-1 analogue, which analogue comprises a first K residue at a position corresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1); a second K residue at a position corresponding to position T of GLP-1(7-37), where T is an integer in the range of 7-37 except 18 and 27; and a maximum of ten amino acid changes as compared to GLP-1(7-37); wherein the first K residue is designated K27, and the second K residue is designated KT; which derivative comprises two albumin binding moieties attached to K27 and KT, respectively, via a linker, wherein the albumin binding moiety comprises a protracting moiety selected from HOOC—(CH2)x—CO— and HOOC—C6H4—O—(CH2)y—CO—; in which x is an integer in the range of 6-16, and y is an integer in the range of 3-17; wherein the linker comprises an element of the formula —NH—(CH2)2—(O—(CH2)2)k—O—(CH2)n—CO—, wherein k is an integer in the range of 1-5, and n is an integer in the range of 1-5; or a pharmaceutically acceptable salt, amide, or ester thereof. The invention also relates to the pharmaceutical use thereof, for example in the treatment and/or prevention of all forms of diabetes and related diseases, as well as to corresponding novel GLP-1 analogues. The derivatives are suitable for oral administration.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/968,950, filed May 2, 2018, which is a continuation of U.S.application Ser. No. 15/346,323, filed Nov. 8, 2016 (Issued U.S. Pat.No. 10,005,827, issued Jun. 26, 2018), which is a continuation of U.S.application Ser. No. 14/961,064, filed Dec. 7, 2015 (Issued U.S. Pat.No. 9,527,900, issued Dec. 27, 2016), which is a continuation of U.S.application Ser. No. 14/009,902, filed Dec. 4, 2013 (Issued U.S. Pat.No. 9,266,940, issued Feb. 23, 2016), which is a 35 U.S.C. § 371National Stage application of International ApplicationPCT/EP2012/056642 (WO 2012/140117), filed Apr. 12, 2012, which claimedpriority of European Patent Application 11162087.8, filed Apr. 12, 2011;and under 35 U.S.C. § 119 of U.S. Provisional Application 61/474,913,filed Apr. 13, 2011; the contents of all above-named applications areincorporated herein by reference.

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 22, 2019, isnamed 8326US05_SeqList_ST25.txt and is 17 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates to derivatives of analogues ofGlucagon-Like Peptide 1 (GLP-1), more in particular to double-acylatedGLP-1 derivatives acylated at K²⁷ and at another K residue of thepeptide, and their pharmaceutical use.

BACKGROUND

Journal of Medicinal Chemistry (2000), vol. 43, no. 9, p. 1664-1669discloses derivatives of GLP-1(7-37) including some that aredouble-acylated.

WO 98/08871 A1 discloses a number of GLP-1 derivatives including somethat are double-acylated. Liraglutide, a mono-acylated GLP-1 derivativefor once daily administration which is marketed as of 2009 by NovoNordisk A/S, is also disclosed in WO 98/08871 A1 (Example 37).

WO 99/43706 A1 discloses a number of mono- and double-acylated GLP-1derivatives including some K^(27,26) and K^(27,34) derivatives.

WO 06/097537 A2 discloses a number of GLP-1 derivatives includingsemaglutide (Example 4), a mono-acylated GLP-1 derivative for onceweekly administration which is under development by Novo Nordisk A/S.

Angewandte Chemie International Edition 2008, vol. 47, p. 3196-3201reports the discovery and characterisation of a class of4-(p-iodophenyl)butyric acid derivatives which purportedly display astable noncovalent binding interaction with both mouse serum albumin(MSA) and human serum albumin (HSA).

SUMMARY

The invention relates to derivatives of GLP-1 peptides.

The derivatives are acylated at a lysine substituted for the nativeglutamic acid at position 27, as well as at another lysine residue. Theside chains are albumin binding moieties. They comprise a protractingmoiety, preferably selected from fatty diacids, and fatty acids with adistal phenoxy group, all optionally substituted. A carboxy group of thefatty acid or fatty diacid is acylated, optionally via a linker, to alysine residue of the GLP-1 peptide, preferably at the epsilon-aminogroup thereof.

The GLP-1 peptide may be an analogue of GLP-1(7-37) (SEQ ID NO: 1)having a total of up to ten amino acid differences as compared toGLP-1(7-37), for example one or more additions, one or more deletions,and/or one or more substitutions.

More in particular, the invention relates to a derivative of a GLP-1analogue, which analogue comprises a first K residue at a positioncorresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1); a second Kresidue at a position corresponding to position T of GLP-1(7-37), whereT is an integer in the range of 7-37 except 18 and 27; and a maximum often amino acid changes as compared to GLP-1(7-37); wherein the first Kresidue is designated K²⁷, and the second K residue is designated K^(T);which derivative comprises two albumin binding moieties attached to K²⁷and K^(T), respectively, via a linker, wherein each albumin bindingmoiety comprises a protracting moiety selected from HOOC—(CH₂)_(x)—CO—*and HOOC—C₆H₄—O—(CH₂)_(y)—CO—*, in which x is an integer in the range of6-16, and y is an integer in the range of 3-17, and wherein the linkercomprises a linker element of formula Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; or a pharmaceutically acceptable salt, amide, or esterthereof.

The invention also relates to such derivative for use as a medicament,in particular for use in the treatment and/or prevention of all forms ofdiabetes and related diseases, such as eating disorders, cardiovasculardiseases, gastrointestinal diseases, diabetic complications, criticalillness, and/or polycystic ovary syndrome; and/or for improving lipidparameters, improving β-cell function, and/or for delaying or preventingdiabetic disease progression.

The invention furthermore relates to intermediate products in the formof novel GLP-1 analogues, which are relevant for the preparation ofcertain derivatives of the invention.

The derivatives of the invention are biologically active. Also, oralternatively, they have a protracted pharmacokinetic profile. Also, oralternatively, they are stable against degradation by gastro intestinalenzymes. Also, or alternatively, they have a high oral bioavailability.These properties are of importance in the development of next generationGLP-1 compounds for subcutaneous, intravenous, and/or in particular oraladministration.

DESCRIPTION

In what follows, Greek letters may be represented by their symbol or thecorresponding written name, for example: α=alpha; β=beta; ε=epsilon;γ=gamma; ω=omega; etc. Also, the Greek letter of t may be represented by“u”, e.g. in μl=ul, or in μM=uM.

An asterisk (*) in a chemical formula designates i) a point ofattachment, ii) a radical, and/or iii) an unshared electron.

In a first aspect, the invention relates to a derivative of a GLP-1analogue, which analogue comprises a first K residue at a positioncorresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1); a second Kresidue at a position corresponding to position T of GLP-1(7-37), whereT is an integer in the range of 7-37 except 18 and 27; and a maximum often amino acid changes as compared to GLP-1(7-37); wherein the first Kresidue is designated K²⁷, and the second K residue is designated K^(T);which derivative comprises two albumin binding moieties attached to K²⁷and K^(T), respectively, via a linker, wherein the albumin bindingmoiety comprises a protracting moiety selected from Chem. 1 and Chem. 2:

HOOC—(CH₂)_(x)—CO—*  Chem. 1:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

in which x is an integer in the range of 6-16, and y is an integer inthe range of 3-17, and the linker comprises Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; or a pharmaceutically acceptable salt, amide, or esterthereof.

GLP-1 Analogues

The term “GLP-1 analogue” or “analogue of GLP-1” as used herein refersto a peptide, or a compound, which is a variant of the humanGlucagon-Like Peptide-1 (GLP-1(7-37)), the sequence of which is includedin the sequence listing as SEQ ID NO: 1. The peptide having the sequenceof SEQ ID NO: 1 may also be designated “native” GLP-1.

In the sequence listing, the first amino acid residue of SEQ ID NO: 1(histidine) is assigned no. 1. However, in what follows—according toestablished practice in the art—this histidine residue is referred to asno. 7, and subsequent amino acid residues are numbered accordingly,ending with glycine no. 37. Therefore, generally, any reference hereinto an amino acid residue number or a position number of the GLP-1(7-37)sequence is to the sequence starting with His at position 7 and endingwith Gly at position 37.

GLP-1 analogues of the derivatives of the invention may be described byreference to i) the number of the amino acid residue in nativeGLP-1(7-37) which corresponds to the amino acid residue which is changed(i.e., the corresponding position in native GLP-1), and to ii) theactual change. The following are non-limiting examples of suitableanalogue nomenclature.

A non-limiting example of a GLP-1 analogue of the derivative of theinvention is an analogue that is changed so as to comprise a firstlysine residue at a position corresponding to position 27 ofGLP-1(7-37), and a second lysine residue at position 12. The amino acidsequence of this analogue is otherwise identical to that of nativeGLP-1, and this analogue may be designated K¹²,K²⁷-GLP-1(7-37). Thisdesignation represents the amino acid sequence of native GLP-1 wherephenylalanine at position 12 has been substituted with lysine, andglutamic acid at position 27 has been substituted with lysine.

The GLP-1 analogue forming part of the derivative of the inventioncomprises a maximum of ten amino acid changes when compared with nativeGLP-1(7-37) (SEQ ID NO: 1). In other words, it is a GLP-1(7-37) peptidein which a number of amino acid residues have been changed when comparedto native GLP-1(7-37) (SEQ ID NO: 1). These changes may represent,independently, one or more amino acid substitutions, additions, and/ordeletions.

The following are non-limiting examples of appropriate analoguenomenclature.

For example, the analogue[Aib8,Lys22,Val25,Arg26,Lys27,His31,Arg34]-GLP-1-(7-37) designates aGLP-1(7-37) peptide which, when compared to native GLP-1, has thefollowing substitutions: Substitution of alanine at position 8 with Aib(α-aminoisobutyric acid), of glycine at position 22 with lysine, ofalanine at position 25 with valine, of lysine at position 26 witharginine, of glutamic acid at position 27 with lysine, of tryptophan atposition 31 with histidine, and of lysine at position 34 with arginine.This analogue may also be briefly designated (8Aib, 22K, 25V, 26R, 27K,31H, 34R).

As another example, the analogue[Aib8,Lys20,Glu22,Arg26,Lys27,Glu30,Gly34]-GLP-1-(7-34) designates aGLP-1(7-37) peptide, which, when compared to native GLP-1, is changed bysubstitution of alanine at position 8 with Aib, substitution of leucineat position 20 with lysine, substitution of glycine at position 22 withglutamic acid, substitution of lysine at position 26 with arginine,substitution of glutamic acid at position 27 with lysine, substitutionof alanine at position 30 with glutamic acid, substitution of lysine atposition 34 with glycine, and by deletion of the C-terminus ofglycine-arginine-glycine at position 35-36-37. This analogue may also bebriefly designated (8Aib, 20K, 22E, 26R, 27K, 30E, 34G, des35-37), wherereference to GLP-1(7-37) is implied, and “des” represents a deletion.

As a still further example, an analogue comprising Glu³⁸ and Gly³⁹refers to a GLP-1(7-37) peptide, which, when compared to native GLP-1,comprises an addition of the dipeptide of (glutamic acid—glycine) to theC-terminus of GLP-1(7-37). This analogue may also briefly be said tocomprise (38E, 39G), where the reference to GLP-1(7-37) is implied.

Analogues “comprising” certain specified changes may comprise furtherchanges, when compared to SEQ ID NO: 1. One example, non-limiting, of ananalogue comprising (38E, 39G) is the peptide part of Chem. 51.

As is apparent from the above examples, amino acid residues may beidentified by their full name, their one-letter code, and/or theirthree-letter code. These three ways are fully equivalent.

The expressions “a position equivalent to” or “corresponding position”may be used to characterise the site of change in a variant GLP-1(7-37)sequence by reference to native GLP-1(7-37) (SEQ ID NO: 1). Equivalentor corresponding positions, as well as the number of changes, are easilydeduced, e.g. by simple handwriting and eyeballing; and/or a standardprotein or peptide alignment program may be used, such as “align” whichis a Needleman-Wunsch alignment. The algorithm is described inNeedleman, S. B. and Wunsch, C. D., (1970), Journal of MolecularBiology, 48: 443-453, and the align program by Myers and W. Miller in“Optimal Alignments in Linear Space” CABIOS (computer applications inthe biosciences) (1988) 4:11-17. For the alignment, the default scoringmatrix BLOSUM50 and the default identity matrix may be used, and thepenalty for the first residue in a gap may be set at −12, or preferablyat −10, and the penalties for additional residues in a gap at −2, orpreferably at −0.5.

An example of such alignment is inserted hereinbelow, in which sequenceno. 1 (SEQ_ID_NO_1) is SEQ ID NO: 1, and sequence no. 2 (SEQ ID NO: 2)(ANALOGUE) is the analogue (22K, 26R, 27K, 30E, 34G, des35-37) thereof:

# Aligned_sequences: 2 # 1: SEQ_ID_NO_1 # 2: ANALOGUE # Matrix:EBLOSUM62 # Gap_penalty: 10.0 # Extend_penalty: 0.5 # # Length: 31# Identity: 23/31 (74.2%) # Similarity: 25/31 (80.6%) # Gaps: 3/31(9.7%) # Score: 117.0 # SEQ_ID_NO_1 1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 31|||||||||||||||.|||::||.|||. ANALOGUE 1 HAEGIFTSDVSSYLEKQAARKFIEWLVG---28 (SEQ ID NO: 2)

In case of non-natural amino acids such as Aib being included in thesequence, these may, for alignment purposes, be replaced with X. Ifdesired, X can later be manually corrected.

The term “peptide”, as e.g. used in the context of the GLP-1 analoguesof the derivatives of the invention, refers to a compound whichcomprises a series of amino acids interconnected by amide (or peptide)bonds.

The peptides of the invention comprise at least five constituent aminoacids connected by peptide bonds. In particular embodiments the peptidecomprises at least 10, preferably at least 15, more preferably at least20, even more preferably at least 25, or most preferably at least 28amino acids.

In particular embodiments, the peptide is composed of at least fiveconstituent amino acids, preferably composed of at least 10, at least15, at least 20, at least 25, or most preferably composed of at least 28amino acids.

In additional particular embodiments, the peptide is a) composed of, orb) consists of, i) 28, ii) 29, iii) 30, iv) 31, v) 32, or vi) 33 aminoacids.

In a still further particular embodiment the peptide consists of aminoacids interconnected by peptide bonds.

Amino acids are molecules containing an amine group and a carboxylicacid group, and, optionally, one or more additional groups, oftenreferred to as a side chain.

The term “amino acid” includes proteogenic amino acids (encoded by thegenetic code, including natural amino acids, and standard amino acids),as well as non-proteogenic (not found in proteins, and/or not coded forin the standard genetic code), and synthetic amino acids. Thus, theamino acids may be selected from the group of proteinogenic amino acids,non-proteinogenic amino acids, and/or synthetic amino acids.

Non-limiting examples of amino acids which are not encoded by thegenetic code are gamma-carboxyglutamate, ornithine, and phosphoserine.Non-limiting examples of synthetic amino acids are the D-isomers of theamino acids such as D-alanine and D-leucine, Aib (α-aminoisobutyricacid), β-alanine, and des-amino-histidine (desH, alternative nameimidazopropionic acid, abbreviated Imp).

In what follows, all amino acids for which the optical isomer is notstated is to be understood to mean the L-isomer (unless otherwisespecified).

The GLP-1 derivatives and analogues of the invention have GLP-1activity. This term refers to the ability to bind to the GLP-1 receptorand initiate a signal transduction pathway resulting in insulinotropicaction or other physiological effects as is known in the art. Forexample, the analogues and derivatives of the invention can be testedfor GLP-1 activity using the assay described in Example 33 herein. TheGLP-1 receptor binding assay described in Example 34 herein may also beused for determining GLP-1 activity (the low HSA experiment).

GLP-1 Derivatives

The term “derivative” as used herein in the context of a GLP-1 peptideor analogue means a chemically modified GLP-1 peptide or analogue, inwhich one or more substituents have been covalently attached to thepeptide. The substituent may also be referred to as a side chain.

In a particular embodiment, the side chain is capable of formingnon-covalent aggregates with albumin, thereby promoting the circulationof the derivative with the blood stream, and also having the effect ofprotracting the time of action of the derivative, due to the fact thatthe aggregate of the GLP-1-derivative and albumin is only slowlydisintegrated to release the active pharmaceutical ingredient. Thus, thesubstituent, or side chain, as a whole is preferably referred to as analbumin binding moiety.

In another particular embodiment the albumin binding moiety comprises aportion which is particularly relevant for the albumin binding andthereby the protraction, which portion may be referred to as aprotracting moiety. The protracting moiety may be at, or near, theopposite end of the albumin binding moiety, relative to its point ofattachment to the peptide.

In a still further particular embodiment the albumin binding moietycomprises a portion inbetween the protracting moiety and the point ofattachment to the peptide, which portion may be referred to as a linker,linker moiety, spacer, or the like. The linker may be optional, andhence in that case the albumin binding moiety may be identical to theprotracting moiety.

In particular embodiments, the albumin binding moiety and/or theprotracting moiety is lipophilic, and/or negatively charged atphysiological pH (7.4).

The albumin binding moiety, the protracting moiety, or the linker may becovalently attached to a lysine residue of the GLP-1 peptide byacylation.

In a preferred embodiment, an active ester of the albumin bindingmoiety, preferably comprising a protracting moiety and a linker, iscovalently linked to an amino group of a lysine residue, preferably theepsilon amino group thereof, under formation of an amide bond (thisprocess being referred to as acylation).

Unless otherwise stated, when reference is made to an acylation of alysine residue, it is understood to be to the epsilon-amino groupthereof.

A derivative comprising two protracting moieties attached to a first anda second K residue (e.g., to K²⁷ and K^(T)) via a linker may be referredto as a derivative which has been acylated twice, double-acylated, ordual acylated at the epsilon-amino groups of the first and second lysineresidues, e.g. at position 27 and T, respectively, of the GLP-1 peptide.

For the present purposes, the terms “albumin binding moiety”,“protracting moiety”, and “linker” may include the unreacted as well asthe reacted forms of these molecules. Whether or not one or the otherform is meant is clear from the context in which the term is used.

In one aspect, each protracting moiety comprises, or consists of, aprotracting moiety independently selected from Chem. 1 and Chem. 2:

HOOC—(CH₂)_(x)—CO—*  Chem. 1:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

in which x is an integer in the range of 6-16, and y is an integer inthe range of 3-17.

In one embodiment, *—(CH₂)_(x)* refers to straight or branched,preferably straight, alkylene in which x is an integer in the range of6-16.

In another embodiment, *—(CH₂)_(y)—* refers to straight or branched,preferably straight, alkylene in which y is an integer in the range of3-17.

The term “fatty acid” refers to aliphatic monocarboxylic acids havingfrom 4 to 28 carbon atoms, it is preferably unbranched, and/or evennumbered, and it may be saturated or unsaturated.

The term “fatty diacid” refers to fatty acids as defined above but withan additional carboxylic acid group in the omega position. Thus, fattydiacids are dicarboxylic acids.

The nomenclature is as is usual in the art, for example in the aboveformulas *—COOH as well as HOOC—* refers to carboxy; *—C₆H₄—* tophenylene; *—CO—*, as well as *—OC—*, to carbonyl (O═C<**); C₆H₅—O—* tophenoxy. In particular embodiments, the aromatics, such as the phenoxy,and the phenylene radicals, may be, independently, ortho, meta, or para.

As explained above, the GLP-1 derivatives of the present invention aredouble-acylated, i.e. two albumin binding moieties are covalentlyattached to the GLP-1 peptide.

In a particular embodiment, the two albumin binding moieties (i.e. theentire side chains) are similar, preferably substantially identical, or,most preferably, identical.

In another particular embodiment, the two protracting moieties aresimilar, preferably substantially identical, or, most preferably,identical.

In a still further particular embodiment, the two linkers are similar,preferably substantially identical, or, most preferably identical.

The term “substantially identical” includes differences from identitywhich are due to formation of one or more salts, esters, and/or amides;preferably formation of one or more salts, methyl esters, and simpleamides; more preferably formation of no more than two salts, methylesters, and/or simple amides; even more preferably formation of no morethan one salt, methyl ester, and/or simple amide; or most preferablyformation of no more than one salt.

In the context of chemical compounds such as the albumin bindingmoieties, protracting moieties, and linkers, similarity and/or identitymay be determined using any suitable computer program and/or algorithmknown in the art.

For example, the similarity of two protracting moieties, two linkers,and/or two entire side chains may suitably be determined using molecularfingerprints. Fingerprints is a mathematical method of representing achemical structure (see e.g. Chemoinformatics: A textbook, JohannGasteiger and Thomas Engel (Eds), Wiley-VCH Verlag, 2003).

Examples of suitable fingerprints include, without limitation, UNITYfingerprints, MDL fingerprints, and/or ECFP fingerprints, such as ECFP_6fingerprints (ECFP stands for extended-connectivity fingerprints).

In particular embodiments, the two protracting moieties, the twolinkers, and/or the two entire side chains are represented as a) ECFP_6fingerprints; b) UNITY fingerprints; and/or c) MDL fingerprints.

The Tanimoto coefficient is preferably used for calculating thesimilarity of the two fingerprints, whether a), b), or c) is used.

In particular embodiments, whether a), b), or c) is used, the twoprotracting moieties, the two linkers, and/or the two entire sidechains, respectively, have a similarity of at least 0.5 (50%);preferably at least 0.6 (60%); more preferably at least 0.7 (70%), or atleast 0.8 (80%); even more preferably at least 0.9 (90%); or mostpreferably at least 0.99 (99%), such as a similarity of 1.0 (100%).

UNITY fingerprints may be calculated using the programme SYBYL(available from Tripos, 1699 South Hanley Road, St. Louis, Mo.63144-2319 USA). ECFP_6 and MDL fingerprints may be calculated using theprogramme Pipeline Pilot (available from Accelrys Inc., 10188 TelesisCourt, Suite 100, San Diego, Calif. 92121, USA).

For more details, see for example J. Chem. Inf. Model. 2008, 48,542-549; J. Chem. Inf. Comput. Sci. 2004, 44, 170-178; J. Med. Chem.2004, 47, 2743-2749; J. Chem. Inf. Model. 2010, 50, 742-754; as well asSciTegic Pipeline Pilot Chemistry Collection: Basic Chemistry UserGuide, March 2008, SciTegic Pipeline Pilot Data Modeling Collection,2008—both from Accelrys Software Inc., San Diego, US.

An example of a similarity calculation is inserted hereinbelow, in whichthe entire side chain of Chem. 66 was compared with a methyl esterthereof, viz. the mono methyl ester of the glutamine linker moiety (Chem66a):

Using a) ECFP_6 fingerprints the similarity is 0.798, using b) UNITYfingerprints the similarity is 0.957; and using MDL fingerprints thesimilarity is 0.905.

In case of two identical side chains (albumin binding moieties) thederivative may be designated symmetrical.

In particular embodiments, the similarity coefficient is at least 0.80,preferably at least 0.85, more preferably at least 0.90, even morepreferably at least 0.95, or most preferably at least 0.99.

Each of the two linkers of the derivative of the invention may comprisethe following first linker element:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5.

In a particular embodiment, when k=1 and n=1, this linker element may bedesignated OEG, or a di-radical of 8-amino-3,6-dioxaoctanic acid, and/orit may be represented by the following formula:

*—NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—CO—*.  Chem. 5a:

In another particular embodiment, each linker of the derivative of theinvention may comprise, independently, a second linker element,preferably a Glu di-radical, such as Chem. 6 and/or Chem. 7:

wherein the Glu di-radical may be included p times, where p is aninteger in the range of 1-2.

Chem. 6 may also be referred to as gamma-Glu, or briefly gGlu, due tothe fact that it is the gamma carboxy group of the amino acid glutamicacid which may here be used for connection to another linker element, orto the epsilon-amino group of lysine. As explained above, the otherlinker element may, for example, be another Glu residue, or an OEGmolecule. The amino group of Glu may in turn form an amide bond with thecarboxy group of the protracting moiety, or with the carboxy group of,e.g., an OEG molecule, if present, or with the gamma-carboxy group of,e.g., another Glu, if present.

Chem. 7 may also be referred to as alpha-Glu, or briefly aGlu, or simplyGlu, due to the fact that it is the alpha carboxy group of the aminoacid glutamic acid which may here be used for connection to anotherlinker element, or to the epsilon-amino group of lysine.

The above structures of Chem. 6 and Chem. 7 cover the L-form, as well asthe D-form of Glu.

The derivatives of the invention may exist in different stereoisomericforms having the same molecular formula and sequence of bonded atoms,but differing only in the three-dimensional orientation of their atomsin space. The stereoisomerism of the exemplified derivatives of theinvention is indicated in the experimental section, in the names as wellas the structures, using standard nomenclature. Unless otherwise statedthe invention relates to all stereoisomeric forms of the claimedderivative.

The concentration in plasma of the GLP-1 derivatives of the inventionmay be determined using any suitable method. For example, LC-MS (LiquidChromatography Mass Spectroscopy) may be used, or immunoassays such asRIA (Radio Immuno Assay), ELISA (Enzyme-Linked Immuno Sorbent Assay),and LOCI (Luminescence Oxygen Channeling Immunoasssay). Generalprotocols for suitable RIA and ELISA assays are found in, e.g.,WO09/030738 on p. 116-118. A preferred assay is the LOCI assay describedin Example 35, 39, and 40 herein.

Pharmaceutically Acceptable Salt, Amide, or Ester

The derivatives and analogues of the invention may be in the form of apharmaceutically acceptable salt, amide, or ester.

Salts are e.g. formed by a chemical reaction between a base and an acid,e.g.: 2 NH₃+H₂SO₄→(NH₄)₂SO₄.

The salt may be a basic salt, an acid salt, or it may be neither nor(i.e. a neutral salt). Basic salts produce hydroxide ions and acid saltshydronium ions in water.

The salts of the derivatives of the invention may be formed with addedcations or anions that react with anionic or cationic groups,respectively. These groups may be situated in the peptide moiety, and/orin the side chain of the derivatives of the invention.

Non-limiting examples of anionic groups of the derivatives of theinvention include free carboxylic groups in the side chain, if any, aswell as in the peptide moiety. The peptide moiety often includes a freecarboxylic acid group at the C-terminus, and it may also include freecarboxylic groups at internal acid amino acid residues such as Asp andGlu.

Non-limiting examples of cationic groups in the peptide moiety includethe free amino group at the N-terminus, if present, as well as any freeamino group of internal basic amino acid residues such as His, Arg, andLys.

The ester of the derivatives of the invention may, e.g., be formed bythe reaction of a free carboxylic acid group with an alcohol or aphenol, which leads to replacement of at least one hydroxyl group by analkoxy or aryloxy group

The ester formation may involve the free carboxylic group at theC-terminus of the peptide, and/or any free carboxylic group in the sidechain.

The amide of the derivatives of the invention may, e.g., be formed bythe reaction of a free carboxylic acid group with an amine or asubstituted amine, or by reaction of a free or substituted amino groupwith a carboxylic acid.

The amide formation may involve the free carboxylic group at theC-terminus of the peptide, any free carboxylic group in the side chain,the free amino group at the N-terminus of the peptide, and/or any freeor substituted amino group of the peptide in the peptide and/or the sidechain.

In a particular embodiment, the peptide or derivative is in the form ofa pharmaceutically acceptable salt. In another particular embodiment,the derivative is in the form of a pharmaceutically acceptable amide,preferably with an amide group at the C-terminus of the peptide. In astill further particular embodiment, the peptide or derivative is in theform a pharmaceutically acceptable ester.

Intermediate Products

In a second aspect, the invention relates to intermediate products.

One type of intermediate product of the invention takes the form of aGLP-1 analogue which comprises the following changes as compared toGLP-1(7-37) (SEQ ID NO: 1): (i) 38Q; and/or (ii) 39G; or apharmaceutically acceptable, salt, amide, or ester thereof.

Another intermediate product of the invention in the form of a GLP-1analogue is an analogue comprising, preferably having, the followingamino acid changes, as compared to GLP-1(7-37) (SEQ ID NO: 1): (i) 22E,26R, 27K, 34R, 37K; (ii) 22E, 26R, 27K, 30E, 34R, 36K, 38E, 39G; (iii)22E, 26R, 27K, 34R, 36K, des37; (iv) 22E, 25V, 26R, 27K, 34R, 37K; (v)8Aib, 20K, 22E, 26R, 27K, 30E, 34G, des35-37; (vi) 26R, 27K, 30E, 34R,36K, 38E; (vii) 8Aib, 22K, 25V, 26R, 27K, 31H, 34R; (iix) 8Aib, 22K,25V, 26R, 27K, 34R, des35-37; (ix) 8Aib, 22K, 25V, 26R, 27K, 34R,des36-37; (x) 26H, 27K, 30E, 34R, 36K, 38E; (xi) 22K, 25V, 26R, 27K,30E, 34Q; (xii) 25V, 26R, 27K, 30E, 34R, 36K, 38Q; (xiii) 25V, 26R, 27K,30E, 34Q, 36K, 38E; (xiv) 22K, 26R, 27K, 31H, 34G, des35-37; (xv) 8Aib,25V, 26R, 27K, 31H, 34Q, 37K; (xvi) 25V, 26R, 27K, 31H, 34Q, 37K; (xvii)22E, 23E, 25V, 26R, 27K, 31H, 34Q, 37K; (iixx) 8Aib, 12K, 22E, 26R, 27K,31H, 34Q; (ixx) 8Aib, 22K, 26R, 27K, 31H, 34G, des35-37; (xx) 22E, 26H,27K, 30E, 34R, 36K, 38E; (xxi) 22E, 24K, 26R, 27K, 31H, 34G, des35-37;(xxii) 25V, 26R, 27K, 34Q, 36K; (xxiii) 22E, 24K, 25V, 26R, 27K, 31H,34R; (xxiv) 22E, 24K, 25V, 26R, 27K, 34G, des35-37; (xxv) 22E, 24K, 25V,26R, 27K, 34R; (xxvi) 8Aib, 22E, 24K, 25V, 26R, 27K, 31H, 34Q; or(xxvii) 8Aib, 22E, 26R, 27K, 30E, 34R, 36K, 38E, 39G; or apharmaceutically acceptable salt, amide, or ester thereof.

Functional Properties

In a first functional aspect, the derivatives of the invention have agood potency. Also, or alternatively, in a second functional aspect,they have a protracted pharmacokinetic profile. Also, or alternatively,in a third functional aspect, they have a high oral bioavailability.Also, or alternatively, in a fourth functional aspect, their biophysicalproperties are improved.

Biological Activity (Potency)

According to the first functional aspect, the derivatives of theinvention, as well as the constituent GLP-1 peptides as such, arebiologically active, or potent.

In a particular embodiment, potency and/or activity refers to in vitropotency, i.e. performance in a functional GLP-1 receptor assay, more inparticular to the capability of stimulating cAMP formation in a cellline expressing the cloned human GLP-1 receptor.

The stimulation of the formation of cAMP in a medium containing thehuman GLP-1 receptor may preferably be determined using a stabletransfected cell-line such as BHK467-12A (tk-ts13), and/or using for thedetermination of cAMP a functional receptor assay, e.g. based oncompetition between endogenously formed cAMP and exogenously addedbiotin-labelled cAMP, in which assay cAMP is more preferably capturedusing a specific antibody, and/or wherein an even more preferred assayis the AlphaScreen cAMP Assay, most preferably the one described inExample 33.

The term half maximal effective concentration (EC₅₀) generally refers tothe concentration which induces a response halfway between the baselineand maximum, by reference to the dose response curve. EC₅₀ is used as ameasure of the potency of a compound and represents the concentrationwhere 50% of its maximal effect is observed.

The in vitro potency of the derivatives of the invention may bedetermined as described above, and the EC₅₀ of the derivative inquestion determined. The lower the EC₅₀, the better the potency.

A suitable medium has the following composition (final in-assayconcentrations): 50 mM TRIS-HCl; 5 mM HEPES; 10 mM MgCl₂, 6H₂O; 150 mMNaCl; 0.01% Tween; 0.1% BSA; 0.5 mM IBMX; 1 mM ATP; 1 uM GTP; pH 7.4.

The EC₅₀ of the derivatives of the invention is at or below 3500 pM,preferably at or below 3200. The EC₅₀ may even be below 1200 pM,preferably below 1000 pM, even more preferably below 500 pM, or mostpreferably below 200 pM.

In another particular embodiment of the first functional aspect, potencyand/or activity refers to the capability of binding to the GLP-1receptor at a low concentration of albumin. The binding to the GLP-1receptor at low albumin concentration should be as good as possible,corresponding to a low IC₅₀ value. This may be determined as describedin Example 35. The IC₅₀ (low albumin) of the derivatives of theinvention is at or below 500 nM, many are below 100 nM, or even below 10nM.

In another particular embodiment the derivatives of the invention arepotent in vivo, which may be determined as is known in the art in anysuitable animal model, as well as in clinical trials.

The diabetic db/db mouse is one example of a suitable animal model, andthe blood glucose lowering effect may be determined in such mice invivo, e.g. as described in Example 36, or as described in Example 43 ofWO09/030738.

Also, or alternatively, the effect on food intake in vivo may bedetermined in pharmacodynamic studies in pigs, e.g. as described inExample 38.

Protraction—Receptor Binding/Low and High Albumin

According to the second functional aspect, the derivatives of theinvention are protracted.

GLP-1 Receptor Binding

In a particular embodiment protraction refers to the ability of thederivatives of the invention to bind to the GLP-1 receptor in thepresence of a low and a high concentration of albumin, respectively,which may be determined as described in Example 34.

Generally, the binding to the GLP-1 receptor at low albuminconcentration should be as good as possible, corresponding to a low IC₅₀value. In one embodiment low albumin refers to 0.005% HSA. In anotherembodiment low albumin refers to 0.001% HSA.

The IC₅₀ value at high albumin concentration is a measure of theinfluence of albumin on the binding of the derivative to the GLP-1receptor. As is known, the GLP-1 derivatives also bind to albumin. Thisis a generally desirable effect, which extends their lifetime in plasma.Therefore, the IC₅₀ value at high albumin will generally be higher thanthe IC₅₀ value at low albumin, corresponding to a reduced binding to theGLP-1 receptor, caused by albumin binding competing with the binding tothe GLP-1 receptor.

A high ratio (IC₅₀ value (high albumin)/IC₅₀ value (low albumin)) maytherefore be taken as an indication that the derivative in questionbinds well to albumin (may have a long half-life), and also per se bindswell to the GLP-1 receptor (the IC₅₀ value (high albumin) is high, andthe IC₅₀ value (low albumin) is low). On the other hand, albumin bindingmay not always be desirable, or the binding to albumin may become toostrong. Therefore, the desirable ranges for IC₅₀ (low albumin), IC₅₀(high albumin)/, and the ratio high/low may vary from compound tocompound, depending on the intended use and the circumstancessurrounding such use, and on other compound properties of potentialinterest.

A suitable assay for determining receptor binding at high and lowalbumin concentration is disclosed in Example 34 herein. The compoundsof the invention have a very good receptor binding affinity (IC₅₀) inthe presence of low albumin. On average the IC₅₀ (low albumin) of thecompounds tested in Example 34 is 14 nM.

Protraction—Half Life In Vivo in Rats

According to the second functional aspect, the derivatives of theinvention are protracted. In a particular embodiment, protraction maysuitably be determined as half-life (T_(1/2)) in vivo in rats after i.v.administration. The half-life in rat is at least 4 hours, and it may beas high as 10 hours or more.

A suitable assay for determining half-life in vivo in rats after i.v.administration is disclosed in Example 39 herein.

Protraction—Half Life In Vivo in Minipigs

According to the second functional aspect, the derivatives of theinvention are protracted. In a particular embodiment protraction may,also or alternatively, be determined as half-life (T_(1/2)) in vivo inminipigs after i.v. administration. The half-life is at least 12 hours,it may be at least 24 hours, at least 36 hours, at least 48 hours, or atleast 60 hours, or even higher.

A suitable assay for determining half-life in vivo in minipigs afteri.v. administration is disclosed in Example 37 herein.

Oral Bioavailability

According to the third functional aspect, the derivatives of theinvention have a high oral bioavailability.

The oral bioavailability of commercial GLP-1 derivatives is very low.The oral bioavailability of GLP-1 derivatives under development for i.v.or s.c. administration is also low.

Accordingly, there is a need in the art for GLP-1 derivatives of animproved oral bioavailability. Such derivatives could be suitablecandidates for oral administration, as long as their potency isgenerally satisfactory, and/or as long as their half-life is alsogenerally satisfactory.

The present inventors identified a novel class of GLP-1 derivatives,which have a surprisingly high oral bioavailability, and at the sametime a satisfactory potency, and/or half-life.

Also, or alternatively, these derivatives have a surprisingly improvedoral bioavailability, and at the same time a high binding affinity (i.e.a low IC₅₀ value) to the GLP-1 receptor at a low concentration ofalbumin.

These features are of importance with a view to obtaining a low dailyoral dose of the active pharmaceutical ingredient, which is desirablefor various reasons, including, e.g., economy of production, likelihoodof potential safety issues, as well as administration comfort issues,and environmental concerns.

Generally, the term bioavailability refers to the fraction of anadministered dose of the active pharmaceutical ingredient (API), such asa derivative of the invention that reaches the systemic circulationunchanged. By definition, when an API is administered intravenously, itsbioavailability is 100%. However, when it is administered via otherroutes (such as orally), its bioavailability decreases (due todegradation and/or incomplete absorption and first-pass metabolism).Knowledge about bioavailability is essential when calculating dosagesfor non-intravenous routes of administration.

Absolute oral bioavailability compares the bioavailability (estimated asthe area under the curve, or AUC) of the API in systemic circulationfollowing oral administration, with the bioavailability of the same APIfollowing intravenous administration. It is the fraction of the APIabsorbed through non-intravenous administration compared with thecorresponding intravenous administration of the same API. The comparisonmust be dose normalised if different doses are used; consequently, eachAUC is corrected by dividing the corresponding dose administered.

A plasma API concentration vs time plot is made after both oral andintravenous administration. The absolute bioavailability (F) is thedose-corrected AUC-oral divided by AUC-intravenous.

The derivatives of the invention have an absolute oral bioavailabilitywhich is higher than that of a) liraglutide, and/or b) semaglutide;preferably at least 10% higher, more preferably at least 20% higher,even more preferably at least 30% higher, or most preferably at least40% higher. Before testing oral bioavailability the derivatives of theinvention may suitably be formulated as is known in the art of oralformulations of insulinotropic compounds, e.g. using any one or more ofthe formulations described in WO 2008/145728.

Suitable tests predictive of oral bioavailability are described inExamples 35 and 40. According to these tests, after direct injection ofthe GLP-1 derivative into the intestinal lumen of rats and/or after oralgavage of rats, the concentration (exposure) thereof in plasma isdetermined, and the subsequent exposure in plasma of the GLP-1derivative is measured,

Biophysical Properties

According to the fourth functional aspect, the derivatives of theinvention have improved biophysical properties. These properties includebut are not limited to physical stability and solubility. Improvedbiophysical properties may be a result of changed oligomeric properties.The biophysical properties may be measured using standard biophysicalmethods of protein chemistry. The biophysical properties of thederivatives of the invention may suitably be compared to those of nativeGLP-1.

Additional particular embodiments of the invention are described in thesection headed “particular embodiments”.

Production Processes

The production of peptides like GLP-1(7-37) and GLP-1 analogues is wellknown in the art.

The GLP-1 moiety of the derivatives of the invention (or fragmentsthereof), such as K¹²,K²⁷-GLP-1(7-37) or an analogue or fragmentthereof, may for instance be produced by classical peptide synthesis,e.g., solid phase peptide synthesis using t-Boc or Fmoc chemistry orother well established techniques, see, e.g., Greene and Wuts,“Protective Groups in Organic Synthesis”, John Wiley & Sons, 1999,Florencio Zaragoza Dörwald, “Organic Synthesis on solid Phase”,Wiley-VCH Verlag GmbH, 2000, and “Fmoc Solid Phase Peptide Synthesis”,Edited by W. C. Chan and P. D. White, Oxford University Press, 2000.

Also, or alternatively, they may be produced by recombinant methods,viz. by culturing a host cell containing a DNA sequence encoding theanalogue and capable of expressing the peptide in a suitable nutrientmedium under conditions permitting the expression of the peptide.Non-limiting examples of host cells suitable for expression of thesepeptides are: Escherichia coli, Saccharomyces cerevisiae, as well asmammalian BHK or CHO cell lines.

Those derivatives of the invention which include non-natural amino acidsand/or a covalently attached N-terminal mono- or dipeptide mimetic maye.g. be produced as described in the experimental part. Or see e.g.,Hodgson et al: “The synthesis of peptides and proteins containingnon-natural amino acids”, Chemical Society Reviews, vol. 33, no. 7(2004), p. 422-430; and WO 2009/083549 A1 entitled “Semi-recombinantpreparation of GLP-1 analogues”.

Specific examples of methods of preparing a number of the derivatives ofthe invention are included in the experimental part.

Pharmaceutical Compositions

Pharmaceutical compositions comprising a derivative of the invention ora pharmaceutically acceptable salt, amide, or ester thereof, and apharmaceutically acceptable excipient may be prepared as is known in theart.

The term “excipient” broadly refers to any component other than theactive therapeutic ingredient(s). The excipient may be an inertsubstance, an inactive substance, and/or a not medicinally activesubstance.

The excipient may serve various purposes, e.g. as a carrier, vehicle,diluent, tablet aid, and/or to improve administration, and/or absorptionof the active substance.

The formulation of pharmaceutically active ingredients with variousexcipients is known in the art, see e.g. Remington: The Science andPractice of Pharmacy (e.g. 19^(th) edition (1995), and any latereditions).

Non-limiting examples of excipients are: Solvents, diluents, buffers,preservatives, tonicity regulating agents, chelating agents, andstabilisers.

Examples of formulations include liquid formulations, i.e. aqueousformulations comprising water. A liquid formulation may be a solution,or a suspension. An aqueous formulation typically comprises at least 50%w/w water, or at least 60%, 70%, 80%, or even at least 90% w/w of water.

Alternatively, a pharmaceutical composition may be a solid formulation,e.g. a freeze-dried or spray-dried composition, which may be used as is,or whereto the physician or the patient adds solvents, and/or diluentsprior to use.

The pH in an aqueous formulation may be anything between pH 3 and pH 10,for example from about 7.0 to about 9.5; or from about 3.0 to about 7.0.

A pharmaceutical composition may comprise a buffer. The buffer may e.g.be selected from the group consisting of sodium acetate, sodiumcarbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine,sodium dihydrogen phosphate, disodium hydrogen phosphate, sodiumphosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malicacid, succinate, maleic acid, fumaric acid, tartaric acid, asparticacid, and mixtures thereof.

A pharmaceutical composition may comprise a preservative. Thepreservative may e.g. be selected from the group consisting of phenol,o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propylp-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate,2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal,bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate,chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride,chlorphenesine (3p-chlorphenoxypropane-1,2-diol), and mixtures thereof.The preservative may be present in a concentration from 0.1 mg/ml to 20mg/ml.

A pharmaceutical composition may comprise an isotonic agent. Theisotonic agent may e.g. be selected from the group consisting of a salt(e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g.glycine, histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400), and mixtures thereof. Any sugar suchas mono-, di-, or polysaccharides, or water-soluble glucans, includingfor example fructose, glucose, mannose, sorbose, xylose, maltose,lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin,alfa and beta HPCD, soluble starch, hydroxyethyl starch andcarboxymethylcellulose-Na may be used. Sugar alcohol is defined as aC₄-C₈ hydrocarbon having at least one —OH group and includes, forexample, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol,and arabitol. In one embodiment, the sugar alcohol additive is mannitol.

A pharmaceutical composition may comprise a chelating agent. Thechelating agent may e.g. be selected from salts ofethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid,and mixtures thereof. A pharmaceutical composition may comprise astabiliser. The stabiliser may e.g. be one or more oxidation inhibitors,aggregation inhibitors, surfactants, and/or one or more proteaseinhibitors. Non-limiting examples of these various kinds of stabilisersare disclosed in the following.

The term “aggregate formation” refers to a physical interaction betweenthe polypeptide molecules resulting in formation of oligomers, which mayremain soluble, or large visible aggregates that precipitate from thesolution. Aggregate formation by a polypeptide during storage of aliquid pharmaceutical composition can adversely affect biologicalactivity of that polypeptide, resulting in loss of therapeutic efficacyof the pharmaceutical composition. Furthermore, aggregate formation maycause other problems such as blockage of tubing, membranes, or pumpswhen the polypeptide-containing pharmaceutical composition isadministered using an infusion system.

A pharmaceutical composition may comprise an amount of an amino acidbase sufficient to decrease aggregate formation of the polypeptideduring storage of the composition. The term “amino acid base” refers toone or more amino acids (such as methionine, histidine, imidazole,arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), oranalogues thereof. Any amino acid may be present either in its free baseform or in its salt form. Any stereoisomer (i.e., L, D, or a mixturethereof) of the amino acid base may be present.

Methionine (or other sulphuric amino acids or amino acid analogous) maybe added to inhibit oxidation of methionine residues to methioninesulfoxide when the polypeptide acting as the therapeutic agent is apolypeptide comprising at least one methionine residue susceptible tosuch oxidation. Any stereoisomer of methionine (L or D) or combinationsthereof can be used.

A pharmaceutical composition may comprise a stabiliser selected from thegroup of high molecular weight polymers or low molecular compounds. Thestabiliser may e.g. be selected from polyethylene glycol (e.g. PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone,carboxy-/hydroxycellulose or derivates thereof (e.g. HPC, HPC-SL, HPC-Land HPMC), cyclodextrins, sulphur-containing substances asmonothioglycerol, thioglycolic acid and 2-methylthioethanol, anddifferent salts (e.g. sodium chloride). A pharmaceutical composition maycomprise additional stabilising agents such as, but not limited to,methionine and EDTA, which protect the polypeptide against methionineoxidation, and a nonionic surfactant, which protects the polypeptideagainst aggregation associated with freeze-thawing or mechanicalshearing.

A pharmaceutical composition may comprise one or more surfactants,preferably a surfactant, at least one surfactant, or two differentsurfactants. The term “surfactant” refers to any molecules or ions thatare comprised of a water-soluble (hydrophilic) part, and a fat-soluble(lipophilic) part. The surfactant may e.g. be selected from the groupconsisting of anionic surfactants, cationic surfactants, nonionicsurfactants, and/or zwitterionic surfactants.

A pharmaceutical composition may comprise one or more proteaseinhibitors, such as, e.g., EDTA (ethylenediamine tetraacetic acid),and/or benzamidineHCl.

Additional, optional, ingredients of a pharmaceutical compositioninclude, e.g., wetting agents, emulsifiers, antioxidants, bulkingagents, metal ions, oily vehicles, proteins (e.g., human serum albumin,gelatine), and/or a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine).

Still further, a pharmaceutical composition may be formulated as isknown in the art of oral formulations of insulinotropic compounds, e.g.using any one or more of the formulations described in WO 2008/145728.

An administered dose may contain from 0.01 mg-100 mg of the derivative,or from 0.01-50 mg, or from 0.01-20 mg, or from 0.01-10 mg of thederivative.

The derivative may be administered in the form of a pharmaceuticalcomposition. It may be administered to a patient in need thereof atseveral sites, for example, at topical sites such as skin or mucosalsites; at sites which bypass absorption such as in an artery, in a vein,or in the heart; and at sites which involve absorption, such as in theskin, under the skin, in a muscle, or in the abdomen.

The route of administration may be, for example, lingual; sublingual;buccal; in the mouth; oral; in the stomach; in the intestine; nasal;pulmonary, such as through the bronchioles, the alveoli, or acombination thereof; parenteral, epidermal; dermal; transdermal;conjunctival; uretal; vaginal; rectal; and/or ocular. A composition maybe an oral composition, and the route of administration is per oral.

A composition may be administered in several dosage forms, for exampleas a solution; a suspension; an emulsion; a microemulsion; multipleemulsions; a foam; a salve; a paste; a plaster; an ointment; a tablet; acoated tablet; a chewing gum; a rinse; a capsule such as hard or softgelatine capsules; a suppositorium; a rectal capsule; drops; a gel; aspray; a powder; an aerosol; an inhalant; eye drops; an ophthalmicointment; an ophthalmic rinse; a vaginal pessary; a vaginal ring; avaginal ointment; an injection solution; an in situ transformingsolution such as in situ gelling, setting, precipitating, and in situcrystallisation; an infusion solution; or as an implant. A compositionmay be a tablet, optionally coated, a capsule, or a chewing gum.

A composition may further be compounded in a drug carrier or drugdelivery system, e.g. in order to improve stability, bioavailability,and/or solubility. In a particular embodiment a composition may beattached to such system through covalent, hydrophobic, and/orelectrostatic interactions. The purpose of such compounding may be,e.g., to decrease adverse effects, achieve chronotherapy, and/orincrease patient compliance.

A composition may also be used in the formulation of controlled,sustained, protracting, retarded, and/or slow release drug deliverysystems.

Parenteral administration may be performed by subcutaneous,intramuscular, intraperitoneal, or intravenous injection by means of asyringe, optionally a pen-like syringe, or by means of an infusion pump.

A composition may be administered nasally in the form of a solution, asuspension, or a powder; or it may be administered pulmonally in theform of a liquid or powder spray.

Transdermal administration is a still further option, e.g. byneedle-free injection, from a patch such as an iontophoretic patch, orvia a transmucosal route, e.g. buccally.

A composition may be a stabilised formulation. The term “stabilisedformulation” refers to a formulation with increased physical and/orchemical stability, preferably both. In general, a formulation must bestable during use and storage (in compliance with recommended use andstorage conditions) until the expiration date is reached.

The term “physical stability” refers to the tendency of the polypeptideto form biologically inactive and/or insoluble aggregates as a result ofexposure to thermo-mechanical stress, and/or interaction withdestabilising interfaces and surfaces (such as hydrophobic surfaces).The physical stability of an aqueous polypeptide formulation may beevaluated by means of visual inspection, and/or by turbiditymeasurements after exposure to mechanical/physical stress (e.g.agitation) at different temperatures for various time periods.Alternatively, the physical stability may be evaluated using aspectroscopic agent or probe of the conformational status of thepolypeptide such as e.g. Thioflavin T or “hydrophobic patch” probes.

The term “chemical stability” refers to chemical (in particularcovalent) changes in the polypeptide structure leading to formation ofchemical degradation products potentially having a reduced biologicalpotency, and/or increased immunogenic effect as compared to the intactpolypeptide. The chemical stability can be evaluated by measuring theamount of chemical degradation products at various time-points afterexposure to different environmental conditions, e.g. by SEC-HPLC, and/orRP-HPLC.

The treatment with a derivative according to the present invention mayalso be combined with one or more additional pharmacologically activesubstances, e.g. selected from antidiabetic agents, antiobesity agents,appetite regulating agents, antihypertensive agents, agents for thetreatment and/or prevention of complications resulting from orassociated with diabetes and agents for the treatment and/or preventionof complications and disorders resulting from or associated withobesity. Examples of these pharmacologically active substances are:Insulin, sulphonylureas, biguanides, meglitinides, glucosidaseinhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV)inhibitors, inhibitors of hepatic enzymes involved in stimulation ofgluconeogenesis and/or glycogenolysis, glucose uptake modulators,compounds modifying the lipid metabolism such as antihyperlipidemicagents as HMG CoA inhibitors (statins), Gastric Inhibitory Polypeptides(GIP analogs), compounds lowering food intake, RXR agonists and agentsacting on the ATP-dependent potassium channel of the β-cells;Cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin,pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide,repaglinide; β-blockers such as alprenolol, atenolol, timolol, pindolol,propranolol and metoprolol, ACE (angiotensin converting enzyme)inhibitors such as benazepril, captopril, enalapril, fosinopril,lisinopril, alatriopril, quinapril and ramipril, calcium channelblockers such as nifedipine, felodipine, nicardipine, isradipine,nimodipine, diltiazem and verapamil, and α-blockers such as doxazosin,urapidil, prazosin and terazosin; CART (cocaine amphetamine regulatedtranscript) agonists, NPY (neuropeptide Y) antagonists, PYY agonists, Y2receptor agonists, Y4 receptor agonits, mixed Y2/Y4 receptor agonists,MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosisfactor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP(corticotropin releasing factor binding protein) antagonists, urocortinagonists, β3 agonists, oxyntomodulin and analogues, MSH(melanocyte-stimulating hormone) agonists, MCH (melanocyte-concentratinghormone) antagonists, CCK (cholecystokinin) agonists, serotoninre-uptake inhibitors, serotonin and noradrenaline re-uptake inhibitors,mixed serotonin and noradrenergic compounds, 5HT (serotonin) agonists,bombesin agonists, galanin antagonists, growth hormone, growth hormonereleasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DAagonists (bromocriptin, doprexin), lipase/amylase inhibitors, RXR(retinoid X receptor) modulators, TR β agonists; histamine H3antagonists, Gastric Inhibitory Polypeptide agonists or antagonists (GIPanalogs), gastrin and gastrin analogs.

The treatment with a derivative according to this invention may also becombined with a surgery that influences the glucose levels, and/or lipidhomeostasis such as gastric banding or gastric bypass.

Pharmaceutical Indications

In a third aspect, the present invention also relates to a derivative ofthe invention, for use as a medicament.

In particular embodiments, the derivative of the invention may be usedfor the following medical treatments, all preferably relating one way orthe other to diabetes:

(i) prevention and/or treatment of all forms of diabetes, such ashyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetesof the young), gestational diabetes, and/or for reduction of HbA1C;

(ii) delaying or preventing diabetic disease progression, such asprogression in type 2 diabetes, delaying the progression of impairedglucose tolerance (IGT) to insulin requiring type 2 diabetes, and/ordelaying the progression of non-insulin requiring type 2 diabetes toinsulin requiring type 2 diabetes;

(iii) improving β-cell function, such as decreasing β-cell apoptosis,increasing β-cell function and/or β-cell mass, and/or for restoringglucose sensitivity to β-cells;

(iv) prevention and/or treatment of cognitive disorders;

(v) prevention and/or treatment of eating disorders, such as obesity,e.g. by decreasing food intake, reducing body weight, suppressingappetite, inducing satiety; treating or preventing binge eatingdisorder, bulimia nervosa, and/or obesity induced by administration ofan antipsychotic or a steroid; reduction of gastric motility; and/ordelaying gastric emptying;

(vi) prevention and/or treatment of diabetic complications, such asneuropathy, including peripheral neuropathy; nephropathy; orretinopathy;

(vii) improving lipid parameters, such as prevention and/or treatment ofdyslipidemia, lowering total serum lipids; lowering HDL; lowering small,dense LDL; lowering VLDL: lowering triglycerides; lowering cholesterol;increasing HDL; lowering plasma levels of lipoprotein a (Lp(a)) in ahuman; inhibiting generation of apolipoprotein a (apo(a)) in vitroand/or in vivo;

(iix) prevention and/or treatment of cardiovascular diseases, such assyndrome X; atherosclerosis; myocardial infarction; coronary heartdisease; stroke, cerebral ischemia; an early cardiac or earlycardiovascular disease, such as left ventricular hypertrophy; coronaryartery disease; essential hypertension; acute hypertensive emergency;cardiomyopathy; heart insufficiency; exercise tolerance; chronic heartfailure; arrhythmia; cardiac dysrhythmia; syncopy; atheroschlerosis;mild chronic heart failure; angina pectoris; cardiac bypass reocclusion;intermittent claudication (atheroschlerosis oblitterens); diastolicdysfunction; and/or systolic dysfunction;

(ix) prevention and/or treatment of gastrointestinal diseases, such asinflammatory bowel syndrome; small bowel syndrome, or Crohn's disease;dyspepsia; and/or gastric ulcers;

(x) prevention and/or treatment of critical illness, such as treatmentof a critically ill patient, a critical illness poly-nephropathy (CIPNP)patient, and/or a potential CIPNP patient; prevention of criticalillness or development of CIPNP; prevention, treatment and/or cure ofsystemic inflammatory response syndrome (SIRS) in a patient; and/or forthe prevention or reduction of the likelihood of a patient sufferingfrom bacteraemia, septicaemia, and/or septic shock duringhospitalisation; and/or

(xi) prevention and/or treatment of polycystic ovary syndrome (PCOS).

In a particular embodiment, the indication is selected from the groupconsisting of (i)-(iii) and (v)-(iix), such as indications (i), (ii),and/or (iii); or indication (v), indication (vi), indication (vii),and/or indication (iix).

In another particular embodiment, the indication is (i). In a furtherparticular embodiment the indication is (v). In a still furtherparticular embodiment the indication is (iix).

The following indications are particularly preferred: Type 2 diabetes,and/or obesity.

Particular Embodiments

The following are particular embodiments of the invention.

1. A derivative of a GLP-1 analogue,

wherein the GLP-1 analogue comprises a first K residue at a positioncorresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1); a second Kresidue at a position corresponding to position T of GLP-1(7-37), whereT is an integer in the range of 7-37 except 18 and 27; and a maximum often amino acid changes as compared to GLP-1(7-37); wherein the first Kresidue is designated K²⁷, and the second K residue is designated K^(T);

which derivative comprises a first and a second protracting moietyattached to K²⁷ and K^(T), respectively, via a first and a secondlinker, respectively, wherein

the first and the second protracting moiety is selected from Chem. 1 andChem. 2:

HOOC—(CH₂)_(x)—CO—*  Chem. 1:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

in which x is an integer in the range of 6-16, y is an integer in therange of 3-17; and

the first and second linker comprises Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5;

or a pharmaceutically acceptable salt, amide, or ester thereof.

2. The derivative of embodiment 1, wherein T is an integer selected fromthe range of 7-37 except 18 and 27.3. The derivative of any of embodiments 1-2, wherein T is selected fromany of the ranges of 7-17, 19-26, and 28-37.4. The derivative of any of embodiments 1-3, wherein T is selected fromthe range of 7-17.5. The derivative of any of embodiments 1-4, wherein T is 12.6. The derivative of any of embodiments 1-3, wherein T is selected fromthe range of 19-26.7. The derivative of any of embodiments 1-3, and 6, wherein T isselected from the group consisting of 20, 22, and 24.8. The derivative of any of embodiments 1-3, and 6-7, wherein T is 20.9. The derivative of any of embodiments 1-3, and 6-7, wherein T is 22 or24.10. The derivative of any of embodiments 1-3, 6-7, and 9, wherein T is22.11. The derivative of any of embodiments 1-3, 6-7, and 9, wherein T is24.12. The derivative of any of embodiments 1-3, wherein T is selected fromthe range of 28-37.13. The derivative of any of embodiments 1-3, and 12, wherein T isselected from the group consisting of 36 and 37.14. The derivative of any of embodiments 1-3, and 12-13, wherein T is36.15. The derivative of any of embodiments 1-3, and 12, wherein T is 37.16. The derivative of any of embodiments 1-16, wherein the positioncorresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1) is identifiedby handwriting and eyeballing.17. The derivative of any of embodiments 1-16, wherein the positioncorresponding to position T of GLP-1(7-37) (SEQ ID NO: 1) is identifiedby handwriting and eyeballing.18. The derivative of any of embodiments 1-17, wherein the positioncorresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1) is identifiedby use of a standard protein or peptide alignment program.19. The derivative of any of embodiments 1-18, wherein the positioncorresponding to position T of GLP-1(7-37) (SEQ ID NO: 1) is identifiedby use of a standard protein or peptide alignment program.20. The derivative of embodiment 19, wherein the alignment program is aNeedleman-Wunsch alignment.21. The derivative of any of embodiments 19-20, wherein the defaultscoring matrix and the default identity matrix is used.22. The derivative of any of embodiments 19-21, wherein the scoringmatrix is BLOSUM62.23. The derivative of any of embodiments 19-22, wherein the penalty forthe first residue in a gap is −10 (minus ten).24. The derivative of any of embodiments 19-23, wherein the penaltiesfor additional residues in a gap is −0.5 (minus point five).25. The derivative of any of embodiments 1-24, wherein the analoguecomprises no K residues other than the first and the second K residue.26. The derivative of any of embodiments 1-25, wherein the protractingmoiety is Chem. 1.27. The derivative of any of embodiments 1-26, wherein x is an evennumber.28. The derivative of any of embodiments 1-27, wherein x is 12.29. The derivative of any of embodiments 1-28, wherein Chem. 1 isrepresented by Chem. 1a:

30. The derivative of any of embodiments 1-25, wherein the protractingmoiety is Chem. 2.31. The derivative of any of embodiments 1-25, and 30, wherein Chem. 2is represented by Chem. 2a:

32. The derivative of any of embodiments 1-25, and 30-31, wherein y isan odd number.33. The derivative of any of embodiments 1-25, and 30-32, wherein y isan integer in the range of 9-11.34. The derivative of any of embodiments 1-25, and 30-33, wherein y is9.35. The derivative of any of embodiments 1-25, and 30-33, wherein y is11.36. The derivative of any of embodiments 1-25, and 30-35, wherein Chem.2 is represented by Chem. 2b, or Chem. 2c:

37. The derivative of any of embodiments 1-25, and 30-35, wherein Chem.2 is represented by Chem. 2b.38. The derivative of any of embodiments 31-35, wherein Chem. 2a isrepresented by Chem. 2b, or Chem. 2c:

39. The derivative of any of embodiments 31-35, and 38, wherein Chem. 2ais represented by Chem. 2b.40. The derivative of any of embodiments 1-39, wherein Chem. 5 is afirst linker element.41. The derivative of any of embodiments 1-40, wherein k is 1.42. The derivative of any of embodiments 1-41, wherein n is 1.43. The derivative of any of embodiments 1-42, wherein Chem. 5 isincluded m times, wherein m is an integer in the range of 1-10.44. The derivative of embodiment 43, wherein m is 2.45. The derivative of any of embodiments 43-44, wherein, when m is not1, the Chem. 5 elements are interconnected via amide bond(s).46. The derivative of any of embodiments 1-45, wherein the linkerfurther comprises a second linker element.47. The derivative of embodiment 46, wherein the second linker elementis a Glu di-radical.48. The derivative of any of embodiments 46-47, wherein the secondlinker element is selected from Chem. 6, and/or Chem. 7:

49. The derivative of embodiment 48, wherein the second linker elementis Chem. 6.50. The derivative of any of embodiments 46-49, wherein the Gludi-radical is included p times, wherein p is an integer in the range of1-2.51. The derivative of embodiment 50, wherein p is 1.52. The derivative of embodiment 50, wherein p is 2.53. The derivative of any of embodiments 46-52, wherein the Gludi-radical is a radical of L-Glu.54. The derivative of any of embodiments 46-53, wherein one or more Gludi-radicals and one or more Chem. 5 elements are interconnected viaamide bond(s).55. The derivative of any of embodiments 46-54, wherein the linkerconsists of m times Chem. 5 and p times the Glu di-radical.56. The derivative of embodiment 55, wherein (m,p) is (2,2) or (2,1).57. The derivative of embodiment 56, wherein (m,p) is (2,1).58. The derivative of any of embodiments 55-57, wherein the m Chem. 5elements and the p Glu di-radicals are interconnected via amide bonds.59. The derivative of any of embodiments 1-58, wherein the linker andthe protracting moiety are interconnected via an amide bond.60. The derivative of any of embodiments 1-59, wherein the linker andthe GLP-1 analogue are interconnected via an amide bond.61. The derivative of any of embodiments 1-60, wherein the linker isattached to the epsilon-amino group of the first or the second Kresidue.62. The derivative of any of embodiments 1-61, wherein the linker hasfrom 5 to 41 C-atoms.63. The derivative of any of embodiments 1-62, wherein the linker has 17or 22 C-atoms.64. The derivative of any of embodiments 1-63, wherein the linker has 17C-atoms.65. The derivative of any of embodiments 1-63, wherein the linker has 22C-atoms.66. The derivative of embodiments 1-65, wherein the linker has from 4 to28 hetero atoms.67. The derivative of any of embodiments 1-66, wherein the linker has 12or 16 hetero atoms.68. The derivative of any of embodiments 1-67, wherein the linker has 12hetero atoms.69. The derivative of any of embodiments 1-67, wherein the linker has 16hetero atoms.70. The derivative of any of embodiments 66-70, wherein the hetero atomsare N-, and/or O-atoms.71. The derivative of any of embodiments 1-70, wherein the linker hasfrom 1 to 7 N-atoms.72. The derivative of any of embodiments 1-71, wherein the linker has 3or 4 N-atoms.73. The derivative of any of embodiments 1-72, wherein the linker has 3N-atoms.74. The derivative of any of embodiments 1-72, wherein the linker has 4N-atoms.75. The derivative of any of embodiments 1-74, wherein the linker hasfrom 3 to 21 O-atoms.76. The derivative of any of embodiments 1-75, wherein the linker has 9or 12 O-atoms.77. The derivative of any of embodiments 1-76, wherein the linker has 9O-atoms.78. The derivative of any of embodiments 1-76, wherein the linker has 12O-atoms.79. The derivative of any of embodiments 1-78, wherein the linkerconsists of two times Chem. 6 and two times Chem. 5, interconnected viaamide bonds and in the sequence indicated, the linker being connected atits *—NH end to the *—CO end of the protracting moiety, and at its *—COend to the epsilon amino group of K²⁷ or K^(T) of the GLP-1 analogue.80. The derivative of any of embodiments 1-78, wherein the linkerconsists of two times Chem. 5 and one time Chem. 6, interconnected viaamide bonds and in the sequence indicated, the linker being connected atits *—NH end to the *—CO end of the protracting moiety, and at its free*—CO end to the epsilon amino group of K²⁷ or K^(T) of the GLP-1analogue.81. The derivative of any of embodiments 1-78, wherein the linkerconsists of one time Chem. 6 and two times Chem. 5, interconnected viaamide bonds and in the sequence indicated, the linker being connected atits *—NH end to the *—CO end of the protracting moiety, and at its *—COend to the epsilon amino group of K²⁷ or K^(T) of the GLP-1 analogue.82. The derivative of any of embodiments 1-78, wherein the linkerconsists of one time Chem. 6, two times Chem. 5, and one time Chem. 6,interconnected via amide bonds and in the sequence indicated, the linkerbeing connected at its *—NH end to the *—CO end of the protractingmoiety, and at its *—CO end to the epsilon amino group of K²⁷ or K^(T)of the GLP-1 analogue.83. The derivative of any of embodiments 1-82, wherein the twoprotracting moieties are substantially identical.84. The derivative of any of embodiments 1-83, wherein the twoprotracting moieties are a) at least 80%, b) at least 85%, c) at least90%, d) at least 95%, or e) at least 99% identical.85. The derivative of any of embodiments 1-83, wherein the twoprotracting moieties have a similarity of a) at least 0.5; b) at least0.6; c) at least 0.7, d) at least 0.8; e) at least 0.9; or f) at least0.99.86. The derivative of any of embodiments 1-85, wherein the twoprotracting moieties have a similarity of 1.0.87. The derivative of any of embodiments 1-86, wherein the two linkersare substantially identical.88. The derivative of any of embodiments 1-87, wherein the two linkershave a similarity of at least 0.5.89. The derivative of any of embodiments 1-88, wherein the two linkershave a similarity of a) at least 0.6; b) at least 0.7, c) at least 0.8;d) at least 0.9; or e) at least 0.99.90. The derivative of any of embodiments 1-89, wherein the two linkershave a similarity of 1.0.91. The derivative of any of embodiments 1-90, wherein the two albuminbinders, such as the two side chains consisting of protracting moietyand linker, are substantially identical.92. The derivative of any of embodiments 1-91, wherein the two albuminbinders, such as the two side chains consisting of protracting moietyand linker, are a) at least 80%, b) at least 85%, c) at least 90%, d) atleast 95%, or e) at least 99% identical.93. The derivative of any of embodiments 1-92, wherein the two albuminbinders, such as the two side chains consisting of protracting moietyand linker, have a similarity of a) at least 0.5; b) at least 0.6; c) atleast 0.7, d) at least 0.8; e) at least 0.9; or f) at least 0.99.94. The derivative of any of embodiments 1-92, wherein the two albuminbinders, such as the two side chains consisting of protracting moietyand linker, have a similarity of 1.0.95. The derivative of any of embodiments 83-94, wherein the two chemicalstructures to be compared are represented as fingerprints.96. The derivative of embodiment 95, wherein the fingerprints are a)ECFP_6 fingerprints; b) UNITY fingerprints; and/or c) MDL fingerprints.97. The derivative of any of embodiments 95-96, wherein the Tanimotocoefficient is preferably used for calculating the similarity, oridentity, of the two fingerprints.98. The derivative of any of embodiments 1-97, wherein the number ofamino acid changes as compared to GLP-1(7-37) (SEQ ID NO: 1) areidentified by handwriting and eyeballing.99. The derivative of any of embodiments 1-98, wherein the number ofamino acid changes as compared to GLP-1(7-37) (SEQ ID NO: 1) areidentified by use of a standard protein or peptide alignment program.100. The derivative of embodiment 99, wherein the alignment program is aNeedleman-Wunsch alignment.101. The derivative of any of embodiments 99-100, wherein the defaultscoring matrix and the default identity matrix is used.102. The derivative of any of embodiments 99-101, wherein the scoringmatrix is BLOSUM62.103. The derivative of any of embodiments 99-102, wherein the penaltyfor the first residue in a gap is −10 (minus ten).104. The derivative of any of embodiments 99-103, wherein the penaltiesfor additional residues in a gap is −0.5 (minus point five).105. The derivative of any of embodiments 1-104, wherein the amino acidchange(s) is (are) at one or more positions corresponding to thefollowing positions in GLP-1(7-37) (SEQ ID NO: 1): 8, 12, 20, 22, 23,24, 25, 26, 27, 30, 31, 34, 35, 36, 37, 38, and 39.106. The derivative of any of embodiments 1-105, wherein the analoguecomprises at least one of the following changes: Aib⁸, K¹², K²⁰, E²² orK²², E²³, K²⁴, V²⁵, R²⁶ or H²⁶, K²⁷, E³⁰, H³¹, G³⁴ or R³⁴ or Q³⁴, Des³⁵,K³⁶ or Des³⁶, K³⁷ or Des³⁷, E³⁸ or Q³⁸, and/or G³⁹.107. The derivative of any of embodiments 1-106, wherein the second Kresidue is K¹², and wherein the analogue, in addition to the change K²⁷,further comprises i) a change selected from G³⁴, R³⁴, and Q³⁴, and ii) achange selected from R²⁶ and H²⁶.108. The derivative of any of embodiments 1-106, wherein the second Kresidue is K²⁰, and wherein the analogue, in addition to the change K²⁷,further comprises i) a change selected from G³⁴, R³⁴, and Q³⁴, and ii) achange selected from R²⁶ and H²⁶.109. The derivative of any of embodiments 1-106, wherein the second Kresidue is K²², and wherein the analogue, in addition to the change K²⁷,further comprises i) a change selected from G³⁴, R³⁴, and Q³⁴, and ii) achange selected from R²⁶ and H²⁶.110. The derivative of any of embodiments 1-106, wherein the second Kresidue is K²⁴, and wherein the analogue, in addition to the change K²⁷,further comprises i) a change selected from G³⁴, R³⁴, and Q³⁴, and ii) achange selected from R²⁶ and H²⁶.111. The derivative of any of embodiments 1-106, wherein the second Kresidue is K³⁶, and wherein the analogue, in addition to the change K²⁷,further comprises i) a change selected from G³⁴, R³⁴, and Q³⁴, and ii) achange selected from R²⁶ and H²⁶.112. The derivative of any of embodiments 1-106, wherein the second Kresidue is K³⁷, and wherein the analogue, in addition to the change K²⁷,further comprises i) a change selected from G³⁴, R³⁴, and Q³⁴, and ii) achange selected from R²⁶ and H²⁶.113. The derivative of any of embodiments 1-112, wherein the analoguecomprises at least one of the following changes: Aib⁸, E²², E²³, V²⁵,E³⁰, H³¹, Des³⁵, Des³⁶, Des³⁷, E³⁸ or Q³⁸, and/or G³⁹.114. The derivative of any of embodiments 1-113, wherein the analoguecomprises Aib⁸.115. The derivative of any of embodiments 1-114, wherein the analoguecomprises E²².116. The derivative of any of embodiments 1-115, wherein the analoguecomprises E²³.117. The derivative of any of embodiments 1-116, wherein the analoguecomprises V²⁵.118. The derivative of any of embodiments 1-117, wherein the analoguecomprises E³⁰.119. The derivative of any of embodiments 1-118, wherein the analoguecomprises H³¹.120. The derivative of any of embodiments 1-119, wherein the analoguecomprises Des³⁷.121. The derivative of embodiment 120, wherein the analogue comprisesDes³⁶.122. The derivative of any of embodiment 121, wherein the analoguecomprises Des³⁵123. The derivative of any of embodiments 1-119, wherein the analoguecomprises E³⁸ or Q³⁸.124. The derivative of embodiment 123, wherein the analogue comprisesQ³⁸.125. The derivative of embodiment 123, wherein the analogue comprisesE³⁸.126. The derivative of any of embodiments 123-125, wherein the analoguecomprises G³⁹.127. The derivative of embodiment 122, which is a derivative ofGLP-1(7-34) (amino acids 1-28 of SEQ ID NO: 1).128. The derivative of embodiment 121, which is a derivative ofGLP-1(7-35) (amino acids 1-29 of SEQ ID NO: 1).129. The derivative of embodiment 120, which is a derivative ofGLP-1(7-36) (amino acids 1-30 of SEQ ID NO: 1).130. The derivative of any of embodiments 1-119, which is a derivativeof GLP-1(7-37) (amino acids 1-31 of SEQ ID NO: 1).131. The derivative of any of embodiments 123-125, which is a derivativeof GLP-1(7-38) (amino acids 1-31 of SEQ ID NO: 1, plus one C-terminallyadded amino acid residue).132 The derivative of any of embodiments 126, which is a derivative ofGLP-1(7-39) (amino acids 1-31 of SEQ ID NO: 1, plus two C-terminallyadded amino acid residues).133. The derivative of any of embodiments 1-132, wherein the analoguehas a maximum of nine amino acid changes.134. The derivative of any of embodiments 1-132, wherein the analoguehas a maximum of eight amino acid changes.135. The derivative of any of embodiments 1-132, wherein the analoguehas a maximum of seven amino acid changes.136. The derivative of any of embodiments 1-132, wherein the analoguehas a maximum of six amino acid changes.137. The derivative of any of embodiments 1-132, wherein the analoguehas a maximum of five amino acid changes.138. The derivative of any of embodiments 1-132, wherein the analoguehas a maximum of four amino acid changes.139. The derivative of any of embodiments 1-132, wherein the analoguehas a maximum of three amino acid changes.140. The derivative of any of embodiments 1-132, wherein the analoguehas a maximum of two amino acid changes.141. The derivative of any of embodiments 1-132, wherein the analoguehas a maximum of one amino acid change.142. The derivative of any of embodiments 1-140, wherein the analoguehas a minimum of one amino acid change.143. The derivative of any of embodiments 1-139, wherein the analoguehas a minimum of two amino acid changes.144. The derivative of any of embodiments 1-138, wherein the analoguehas a minimum of three amino acid changes.145. The derivative of any of embodiments 1-137, wherein the analoguehas a minimum of four amino acid changes.146. The derivative of any of embodiments 1-136, wherein the analoguehas a minimum of five amino acid changes.147. The derivative of any of embodiments 1-135, wherein the analoguehas a minimum of six amino acid changes.148. The derivative of any of embodiments 1-134, wherein the analoguehas a minimum of seven amino acid changes.149. The derivative of any of embodiments 1-133, wherein the analoguehas a minimum of eight amino acid changes.150. The derivative of any of embodiments 1-132, wherein the analoguehas a minimum of nine amino acid changes.151. The derivative of any of embodiments 1-132, wherein the analoguehas one amino acid change.152. The derivative of any of embodiments 1-132, wherein the analoguehas two amino acid changes.153. The derivative of any of embodiments 1-132, wherein the analoguehas three amino acid changes.154. The derivative of any of embodiments 1-132, wherein the analoguehas four amino acid changes.155. The derivative of any of embodiments 1-132, wherein the analoguehas five amino acid changes.156. The derivative of any of embodiments 1-132, wherein the analoguehas six amino acid changes.157. The derivative of any of embodiments 1-132, wherein the analoguehas seven amino acid changes.158. The derivative of any of embodiments 1-132, wherein the analoguehas eight amino acid changes.159. The derivative of any of embodiments 1-132, wherein the analoguehas nine amino acid changes.160. The derivative of any of embodiments 1-132, wherein the analoguehas ten amino acid changes.161. The derivative of any of embodiments 1-160, wherein the change(s)is (are), independently, substitutions, additions, and/or deletions.162. The derivative of any of embodiments 1-161, wherein the analoguecomprises a GLP-1 analogue of Formula I (SEQ ID NO: 3):

Xaa₇-Xaa₈-Glu-Gly-Thr-Xaa₁₂-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Xaa₂₄-Xaa₂₅-Xaa₂₆-Lys-Phe-Ile-Xaa₃₀-Xaa₃₁-Leu-Val-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉,wherein  Formula I:

Xaa₇ is L-histidine, imidazopropionyl, α-hydroxy-histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, N^(α)-acetyl-histidine, N^(α)-formyl-histidine,α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine,2-pyridylalanine, or 4-pyridylalanine;

Xaa₈ is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or(1-aminocyclooctyl) carboxylic acid;

Xaa₁₂ is Lys or Phe;

Xaa₁₆ is Val or Leu;

Xaa₁₈ is Ser, Arg, Asn, Gin, or Glu;

Xaa₁₉ is Tyr or Gin;

Xaa₂₀ is Leu, Lys, or Met;

Xaa₂₂ is Gly, Glu, Lys, or Aib;

Xaa₂₃ is Gin, Glu, or Arg;

Xaa₂₄ is Ala or Lys;

Xaa₂₅ is Ala or Val;

Xaa₂₆ is Val, His, or Arg;

Xaa₃₀ is Ala, Glu, or Arg;

Xaa₃₁ is Trp or His;

Xaa₃₄ is Glu, Asn, Gly, Gin, or Arg;

Xaa₃₅ is Gly, Aib, or absent;

Xaa₃₆ is Arg, Gly, Lys, or absent;

Xaa₃₇ is Gly, Ala, Glu, Pro, Lys, Arg, or absent;

Xaa₃₈ is Ser, Gly, Ala, Glu, Gin, Pro, Arg, or absent; and

Xaa₃₉ is Gly or absent.

163. The derivative of embodiment 162, wherein the analogue is a GLP-1analogue of Formula I (SEQ ID NO: 3).164. The derivative of any of embodiments 162-163, wherein the peptideof Formula I (SEQ ID NO: 3) is an analogue of GLP-1(7-37) (SEQ ID NO:1).165. The derivative of any of embodiments 162-164, wherein if Xaa₃₈ isabsent, then Xaa₃₉ is also absent.166. The derivative of any of embodiments 162-165, wherein if Xaa₃₇ isabsent, then Xaa₃₈ and Xaa₃₉ are also absent.167. The derivative of any of embodiments 162-166, wherein if Xaa₃₆ isabsent, then Xaa₃₇, Xaa₃₈, and Xaa₃₉ are also absent.168. The derivative of any of embodiments 162-167, wherein if Xaa₃₅ isabsent, then Xaa₃₆, Xaa₃₇, Xaa₃₈, and Xaa₃₉ are also absent.169. The derivative of any of embodiments 162-168, wherein Xaa₇ is His;Xaa₈ is Ala or Aib; Xaa₁₂ is Lys or Phe; Xaa₁₆ is Val; Xaa₁₈ is Ser;Xaa₁₉ is Tyr; Xaa₂₀ is Leu or Lys; Xaa₂₂ is Glu, Gly or Lys; Xaa₂₃ isGin or Glu; Xaa₂₄ is Ala or Lys; Xaa₂₅ is Ala or Val; Xaa₂₆ is His orArg; Xaa₃₀ is Ala or Glu; Xaa₃₁ is Trp or His; Xaa₃₄ is Gly, Gin, orArg; Xaa₃₅ is Gly or absent; Xaa₃₆ is Arg, Lys, or absent; Xaa₃₇ is Gly,Lys, or absent; Xaa₃₈ is Glu or Gin; and Xaa₃₉ is Gly or absent.170. The derivative of any of embodiments 162-169, wherein Xaa₇ is His.171. The derivative of any of embodiments 162-170, wherein Xaa₈ is Ala.172. The derivative of any of embodiments 162-170, wherein Xaa₈ is Aib.173. The derivative of any of embodiments 162-172, wherein Xaa₁₂ is Lys.174. The derivative of any of embodiments 162-172, wherein Xaa₁₂ is Phe.175. The derivative of any of embodiments 162-174, wherein Xaa₁₆ is Val.176. The derivative of any of embodiments 162-175, wherein Xaa₁₈ is Ser.177. The derivative of any of embodiments 162-176, wherein Xaa₁₉ is Tyr.178. The derivative of any of embodiments 162-177, wherein Xaa₂₀ is Leu.179. The derivative of any of embodiments 162-177, wherein Xaa₂₀ is Lys.180. The derivative of any of embodiments 162-179, wherein Xaa₂₂ is Glu.181. The derivative of any of embodiments 162-179, wherein Xaa₂₂ is Gly.182. The derivative of any of embodiments 162-179, wherein Xaa₂₂ is Lys.183. The derivative of any of embodiments 162-182, wherein Xaa₂₃ is Gin.184. The derivative of any of embodiments 162-182, wherein Xaa₂₃ is Glu.185. The derivative of any of embodiments 162-184, wherein Xaa₂₄ is Ala.186. The derivative of any of embodiments 162-184, wherein Xaa₂₄ is Lys.187. The derivative of any of embodiments 162-186, wherein Xaa₂₅ is Ala.188. The derivative of any of embodiments 162-186, wherein Xaa₂₅ is Val.189. The derivative of any of embodiments 162-188, wherein Xaa₂₆ is His.190. The derivative of any of embodiments 162-188, wherein Xaa₂₆ is Arg.191. The derivative of any of embodiments 162-190, wherein Xaa₃₀ is Ala.192. The derivative of any of embodiments 162-190, wherein Xaa₃₀ is Glu.193. The derivative of any of embodiments 162-192, wherein Xaa₃₁ is Trp.194. The derivative of any of embodiments 162-192, wherein Xaa₃₁ is His.195. The derivative of any of embodiments 162-194, wherein Xaa₃₄ is Gly.196. The derivative of any of embodiments 162-194, wherein Xaa₃₄ is Gln.197. The derivative of any of embodiments 162-194, wherein Xaa₃₄ is Arg.198. The derivative of any of embodiments 162-197, wherein Xaa₃₅ is Gly.199. The derivative of any of embodiments 162-198, wherein Xaa₃₅ isabsent.200. The derivative of any of embodiments 162-199, wherein Xaa₃₆ is Arg.201. The derivative of any of embodiments 162-199, wherein Xaa₃₆ is Lys.202. The derivative of any of embodiments 162-199, wherein Xaa₃₆ isabsent.203. The derivative of any of embodiments 162-202, wherein Xaa₃₇ is Gly.204. The derivative of any of embodiments 162-202, wherein Xaa₃₇ is Lys.205. The derivative of any of embodiments 162-202, wherein Xaa₃₇ isabsent.206. The derivative of any of embodiments 162-205, wherein Xaa₃₈ is Glu.207. The derivative of any of embodiments 162-205, wherein Xaa₃₈ is Gin.208. The derivative of any of embodiments 162-205, wherein Xaa₃₈ isabsent.209. The derivative of any of embodiments 162-208, wherein Xaa₃₉ is Gly.210. The derivative of any of embodiments 162-208, wherein Xaa₃₉ isabsent.211. The derivative of any of embodiments 1-210, wherein the analoguecomprises the following amino acid changes, as compared to GLP-1(7-37)(SEQ ID NO: 1): (i) 22E, 26R, 27K, 34R, 37K; (ii) 22E, 26R, 27K, 30E,34R, 36K, 38E, 39G; (iii) 22E, 26R, 27K, 34R, 36K, des37; (iv) 22E, 25V,26R, 27K, 34R, 37K; (v) 8Aib, 20K, 22E, 26R, 27K, 30E, 34G, des35-37;(vi) 26R, 27K, 30E, 34R, 36K, 38E; (vii) 8Aib, 22K, 25V, 26R, 27K, 31H,34R; (iix) 8Aib, 22K, 25V, 26R, 27K, 34R, des35-37; (ix) 8Aib, 22K, 25V,26R, 27K, 34R, des36-37; (x) 26H, 27K, 30E, 34R, 36K, 38E; (xi) 22K,25V, 26R, 27K, 30E, 34Q; (xii) 25V, 26R, 27K, 30E, 34R, 36K, 38Q; (xiii)25V, 26R, 27K, 30E, 34Q, 36K, 38E; (xiv) 22K, 26R, 27K, 31H, 34G,des35-37; (xv) 8Aib, 25V, 26R, 27K, 31H, 34Q, 37K; (xvi) 25V, 26R, 27K,31H, 34Q, 37K; (xvii) 22E, 23E, 25V, 26R, 27K, 31H, 34Q, 37K; (iixx)8Aib, 12K, 22E, 26R, 27K, 31H, 34Q; (ixx) 8Aib, 22K, 26R, 27K, 31H, 34G,des35-37; (xx) 22E, 26H, 27K, 30E, 34R, 36K, 38E; (xxi) 22E, 24K, 26R,27K, 31H, 34G, des35-37; (xxii) 25V, 26R, 27K, 34Q, 36K; (xxiii) 22E,24K, 25V, 26R, 27K, 31H, 34R; (xxiv) 22E, 24K, 25V, 26R, 27K, 34G,des35-37; (xxv) 22E, 24K, 25V, 26R, 27K, 34R; (xxvi) 8Aib, 22E, 24K,25V, 26R, 27K, 31H, 34Q; or (xxvii) 8Aib, 22E, 26R, 27K, 30E, 34R, 36K,38E, 39G.212. The derivative of embodiment 211, wherein the analogue has a set ofamino acid changes as defined in any of (i)-(xxvii).213. A compound selected from the following: Chem. 50, Chem. 51, Chem.52, Chem. 53, Chem. 54, Chem. 55, Chem. 56, Chem. 57, Chem. 58, Chem.59, Chem. 60, Chem. 61, Chem. 62, Chem. 63, Chem. 64, Chem. 65, Chem.66, Chem. 67, Chem. 68, Chem. 69, Chem. 70, Chem. 71, Chem. 72, Chem.73, Chem. 74, Chem. 75, Chem. 76, Chem. 77, Chem. 78, Chem. 79, Chem.80, and Chem. 81; or a pharmaceutically acceptable salt, amide, or esterthereof.214. The compound of embodiment 213 which is a compound according to anyof embodiments 1-212.215. A compound characterised by its name, and selected from a listingof each of the names of the compounds of Examples 1-32 herein, or apharmaceutically acceptable salt, amide, or ester thereof.216. The compound of embodiment 215 which is a compound according to anyof embodiments 1-214.217. The derivative of any of embodiments 1-216, which has GLP-1activity.218. The derivative of embodiment 217, wherein GLP-1 activity refers tothe capability of activating the human GLP-1 receptor.219. The derivative of embodiment 217, wherein activation of the humanGLP-1 receptor is measured in an in vitro assay.220. The derivative of any of embodiments 217-219, wherein activation ofthe human GLP-1 receptor is measured as the potency of cAMP production.221. The derivative of any of embodiments 217-220, which has a potencycorresponding to an EC₅₀a) below 10000 pM, more preferably below 5000 pM, even more preferablybelow 4000 pM, or most preferably below 3000 pM;b) below 2000 pM, preferably below 1500 pM, more preferably below 1200pM, even more preferably below 1000 pM, or most preferably below 500 pM;c) below 400 pM, preferably below 300 pM, more preferably below 200 pM,even more preferably below 150 pM, or most preferably below 100 pM; ord) below 80 pM, preferably below 60 pM, more preferably below 40 pM,even more preferably below 30 pM, or most preferably below 20 pM.222. The derivative of any of embodiments 217-221, wherein the potencyis determined as EC₅₀ for the dose-response curve showing dose-dependentformation of cAMP in a medium containing the human GLP-1 receptor.223. The derivative of any of embodiments 219-222, wherein a stabletransfected cell-line such as BHK467-12A (tk-ts13).224. The derivative of any of embodiments 219-223, wherein for thedetermination of cAMP a functional receptor assay.225. The derivative of any of embodiments 219-224, wherein the assay isbased on competition between endogenously formed cAMP and exogenouslyadded biotin-labelled cAMP.226. The derivative of any of embodiments 219-225, in which assay cAMPis captured using a specific antibody.227. The derivative of any of embodiments 219-226, wherein the assay isthe AlphaScreen cAMP Assay.228. The derivative of any of embodiments 219-227, wherein the assay isdescribed in Example 33.229. The derivative of any of embodiments 217-228, wherein activation ofthe human GLP-1 receptor is measured as the capability of binding to thereceptor in the presence of a low albumin concentration, wherein the lowalbumin concentration is 0.005% HSA, or, preferably, 0.001% HSA.230. The derivative of any of embodiment 217-229, for which the ratio[GLP-1 receptor binding affinity (IC₅₀) in the presence of 2.0% HSA(high albumin), divided by GLP-1 receptor binding affinity (IC₅₀) in thepresence of 0.001% HSA (low albumin)] is:a) at least 1.0, more preferably at least 10, even more preferably atleast 25, or most preferably at least 50;b) at least 60, preferably at least 70, more preferably at least 80,even more preferably at least 90, or most preferably at least 100;c) at least 125, preferably at least 150, more preferably at least 200,still more preferably at least 250, even more preferably at least 400,or most preferably at least 500; ord) at least 600, preferably at least 800, even more preferably at least900, or most preferably at least 1000.231. The derivative of any of embodiments 217-230, for which the GLP-1receptor binding affinity (IC₅₀) in the presence of 0.001% HSA (lowalbumin) isa) below 1000 nM, preferably below 750 nM, more preferably below 500 nM,or most preferably below 400 nM; orb) below 300 nM, preferably below 250 nM, more preferably below 200 nM,or most preferably below 100 nM; orc) below 50.0 nM, preferably below 15.0 nM, more preferably below 10.0nM, even more preferably below 5.0 nM, or most preferably below 1.0 nMd) below 0.80 nM, preferably below 0.60 nM, more preferably below 0.40nM, even more preferably below 0.30 nM, or most preferably below 0.20nM.232. The derivative of embodiments 217-231, for which the GLP-1 receptorbinding affinity (IC₅₀) in the presence of 2.0% HSA (high albumin) isa) below 1000 nM, more preferably below 900 nM, or most preferably below800 nM; or b) below 500 nM, preferably below 400 nM, more preferablybelow 300 nM, even more preferably below 150 nM, or most preferablybelow 50.0 nM.166. The derivative of any of embodiments 217-232, wherein the bindingaffinity to the GLP-1 receptor is measured by way of displacement of¹²⁵I-GLP-1 from the receptor.233. The derivative of any of embodiments 217-232, wherein a SPA bindingassay is used.234. The derivative of any of embodiments 217-233, wherein the GLP-1receptor is prepared using a stable, transfected cell line.235. The derivative of any of embodiments 217-234, wherein a hamstercell line is used, preferably a baby hamster kidney cell line, such asBHK tk-ts13.236. The derivative of any of embodiments 229-235, wherein the IC₅₀value is determined as the concentration which displaces 50% of¹²⁵I-GLP-1 from the receptor.237. The derivative of any of embodiments 1-236, which has an oralbioavailability, preferably an absolute oral bioavailability, which ishigher than that of semaglutide.238. The derivative of embodiment 237, wherein oral bioavailability ismeasured in vivo in rats.239. The derivative of any of embodiments 237-239, wherein oralbioavailability is measured as exposure in plasma after direct injectioninto the intestinal lumen.240. The derivative of any of embodiments 237-239, for which the plasmaconcentration (pM) of the derivative, determined 30 minutes afterinjection of a solution of the derivative in the jejunum of rat, dividedby the concentration (pM) of the injected solution (dose-correctedexposure at 30 min) is a) at least 39, b) at least 40; c) at least 60;d) at least 80; e) at least 100; f) at least 125; or g) at least 150.241. The derivative of any of embodiments 237-240, for which the plasmaconcentration (pM) of the derivative, determined 30 minutes afterinjection of a solution of the derivative in the jejunum of rat, dividedby the concentration (pM) of the injected solution (dose-correctedexposure at 30 min) is a) at least 160, b) at least 180, c) at least200, or d) at least 250.242. The derivative of any of embodiments 237-241, wherein the GLP-1derivative is tested in a concentration of 1000 uM in admixture with 55mg/ml sodium caprate.243. The derivative of any of embodiments 237-242, wherein male SpragueDawley rats are used.244. The derivative of any of embodiments 237-243, wherein the rats havea body weight upon arrival of approximately 240 g.245. The derivative of any of embodiments 237-244, wherein the rats arefasted for approximately 18 hours before the experiment.246. The derivative of any of embodiments 237-245, wherein the rats aretaken into general anaesthesia after having fasted and before theinjection of the derivative in the jejunum.247. The derivative of any of embodiments 237-246, wherein thederivative is administered in the proximal part of the jejunum (10 cmdistal for the duodenum), or in the mid-intestine (50 cm proximal forthe cecum).248. The derivative of any of embodiments 237-247, wherein 100 μl of thederivative is injected into the jejunal lumen through a catheter with asyringe, and subsequently 200 μl of air is pushed into the jejunal lumenwith another syringe, which is then left connected to the catheter toprevent flow back into the catheter.249. The derivative of any of embodiments 237-248, wherein blood samples(200 ul) are collected into EDTA tubes from the tail vein at desiredintervals, such as at times 0, 10, 30, 60, 120 and 240 min, andcentrifuged 5 minutes, 10000 G, at 4° C. within 20 minutes.250. The derivative of any of embodiments 237-249, wherein plasma (e.g.75 ul) is separated, immediately frozen, and kept at −20° C. untilanalyzed for plasma concentration of the derivative.251. The derivative of any of embodiments 237-250, wherein LOCI(Luminescent Oxygen Channeling Immunoassay) is used for analyzing theplasma concentration of the derivative.252. The derivative of any of embodiments 1-251, wherein the derivativeis effective at lowering blood glucose in vivo in db/db mice.253. The derivative of any of embodiments 1-252, wherein the derivativeis effective at lowering body weight in vivo in db/db mice.254. The derivative of any of embodiments 252-253, wherein db/db miceare treated, s.c., with a suitable range of doses of the GLP-1derivative, and blood glucose and/or bodyweight is/are determined atappropriate intervals.255. The derivative of any of embodiments 252-254, wherein the dose ofthe GLP-1 derivative is 0.3 nmol/kg, 1.0 nmol/kg, 3.0 nmol/kg, 10nmol/kg, 30 nmol/kg, and 100 nmol/kg, wherein kg refers to the bodyweight of the mouse.256. The derivative of any of embodiments 252-255, wherein a controlgroup is treated with vehicle, s.c., preferably the medium in which theGLP-1 derivative is dissolved, e.g. with the following composition: 50mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4.257. The derivative of any of embodiments 252-256, wherein blood glucoseis determined, and/or the mice are weighed, at time −½ h (half an hourprior to dosing (t=0)), and at times 1, 2, 4, and 8 h.258. The derivative of any of embodiments 252-257, wherein the glucoseconcentration is measured using the glucose oxidase method.259. The derivative of any of embodiments 252-258, wherein

(i) ED₅₀ (body weight (BW)) is calculated as the dose giving rise tohalf-maximum effect on delta (e.g., decrease) BW 8 hours following thesubcutaneous administration of the derivative; and/or

(ii) ED₅₀ (blood glucose (BG)) is calculated as the dose giving rise tohalf-maximum effect on AUC (Area Under the Curve) delta (e.g., decrease)BG 8 hours and/or 24 hours following the subcutaneous administration ofthe derivative.

260. The derivative of any of embodiments 252-259, wherein a sigmoidaldose-response relationship exists, preferably with a clear definition ofthe maximum response.261. The derivative of any of embodiments 1-260, which has a moreprotracted profile of action than liraglutide.262. The derivative of embodiment 261, wherein protraction meanshalf-life in vivo in a relevant animal species.263. The derivative of any of embodiments 261-262, wherein the animal isa) db/db mice, b) rat, c) pig, and/or, d) minipig.264. The derivative of embodiment 263, wherein the animal is minipig.265. The derivative of any of embodiments 261-264, wherein thederivative is administered i) s.c., and/or, ii) i.v.266. The derivative of any of embodiments 1-265, wherein the derivativeis administered i.v.267. The derivative of any of embodiments 1-266, wherein the terminalhalf-life (T ) after i.v. administration in minipigs isa) at least 12 hours, preferably at least 24 hours, more preferably atleast 36 hours, even more preferably at least 48 hours, or mostpreferably at least 60 hours;b) at least 7 hours, preferably at least 16 hours, more preferably atleast 24 hours, even more preferably at least 30 hours, or mostpreferably at least 40 hours;c) at least 50 hours, preferably at least 60 hours, more preferably atleast 70 hours, even more preferably at least 80 hours, or mostpreferably at least 90 hours.268. The derivative of any of embodiments 264-267, wherein the minipigsare male Göttingen minipigs.269. The derivative of any of embodiments 267-268, wherein the minipigsare 7-14 months of age.270. The derivative of any of embodiments 267-269, wherein the weight ofthe minipigs is 16-35 kg.271. The derivative of any of embodiments 267-270, wherein the minipigsare housed individually, and fed once or twice daily, preferably withSDS minipig diet.272. The derivative of any of embodiments 267-271, wherein thederivative is dosed, i.v., after at least 2 weeks of acclimatisation.273. The derivative of any of embodiments 267-272, wherein the animalsare fasted for approximately 18 h before dosing and for at least 4 hafter dosing, and have ad libitum access to water during the wholeperiod.274. The derivative of any of embodiments 267-273, wherein the GLP-1derivative is dissolved in 50 mM sodium phosphate, 145 mM sodiumchloride, 0.05% tween 80, pH 7.4 to a suitable concentration, preferablyfrom 20-60 nmol/ml.275. The derivative of any of embodiments 267-275, wherein intravenousinjections of the derivative are given in a volume corresponding to 1-2nmol/kg.276. The derivative of any of embodiments 1-275, which causes a reducedfood intake in pigs.277. The derivative of embodiment 276, wherein the intake is reducedrelative to a control, that is preferably vehicle-treated, or untreated.278. The derivative of any of embodiments 276-277, wherein the foodintake (0-24 h) isa) 90% or lower relative to the vehicle-treated control, b) preferably80% or lower, c) more preferably 70% or lower, d) even more preferably60% or lower, or e) most preferably 50% or lower.279. The derivative of any of embodiments 276-278, wherein food intake(0-24 h) refers to the first 24 hours after administration of thederivative or vehicle.280. The derivative of any of embodiments 276-279, wherein the pigs arefemale Landrace Yorkshire Duroc (LYD) pigs.281. The derivative of any of embodiments 276-280, wherein the pigs are3 months of age.282. The derivative if any of embodiments 276-281, wherein the pigs havea weight of 30-35 kg.283. The derivative of any of embodiments 276-282, wherein the animalsare housed in a group for 1-2 weeks for acclimatisation.284. The derivative of any of embodiments 276-283, wherein during theexperimental period the animals are placed in individual pens fromMonday morning to Friday afternoon for measurement of individual foodintake.285. The derivative of any of embodiments 276-284, wherein the animalsare fed ad libitum with pig fodder (such as Svinefoder, Antonio).286. The derivative of any of embodiments 276-285, wherein food intakeis monitored on line by logging the weight of fodder every 15 minutes,preferably using the Mpigwin system.287. The derivative of any of embodiments 276-286, which is dosed 0.3,1.0, 3.0, 10, or 30 nmol/kg.288. The derivative of any of embodiments 276-287, which is dissolved ina phosphate buffer (50 mM phosphate, 145 mM sodium chloride, 0.05% tween80, pH 8), preferably at concentrations of 12, 40, 120, 400, or 1200nmol/ml.289. The derivative of any of embodiments 276-288, wherein the phosphatebuffer serves as vehicle.290. The derivative of any of embodiments 276-289, wherein the animalsare dosed with a single subcutaneous dose of the derivative, or vehicle(preferably with a dose volume of 0.025 ml/kg), on the morning of day 1,and food intake is measured for 4 days after dosing.291. The derivative of any of embodiments 1-290, which has a half-life(T ) in vivo in rats after i.v. administration of a) at least 4 hours,b) at least 6 hours, c) at least 8 hours, or d) at least 10 hours.292. The derivative of any of embodiments 1-291, which has a half-life(T ) in vivo in rats after i.v. administration of a) at least 12 hours,b) at least 15 hours, c) at least 18 hours, or d) at least 20 hours.293. The derivative of any of embodiments 1-292, which has a half-life(T ) in vivo in rats after i.v. administration of a) at least 24 hours,b) at least 26 hours, or c) at least 30 hours.294. The derivative of any of embodiments 291-294, in which the rats aremale Sprague Dawley rats with a body weight of approximately 400 g.294. The derivative of any of embodiments 238-294, for which the AUC ofthe dose-corrected (i.e., divided by the dose in pmol of injectedderivative) plasma exposure curve (i.e., concentration in plasma in pMvs time) from time 30 to 180 min is determined (i.e., the result isindicated in (min x pM/pmol) or simply in min/L).295. The derivative of embodiment 294, wherein the AUC of thedose-corrected plasma exposure curve isa) at least 50, preferably at least 100, or more preferably at least 150min/L;b) at least 200, preferably at least 250, more preferably at least 300,or most preferably at least 320 min/L; orc) at least 1.5 times, preferably at least 2 times, more preferably atleast 3 times, or most preferably at least 4 times the corresponding AUCvalue for semaglutide.296. The derivative of any of embodiments 1-295, wherein oralbioavailability is measured in vivo in rats, as exposure in plasma afteroral gavage.297. The derivative of embodiment 296, for which the AUC of thedose-corrected (i.e., divided by the dose in pmol of administeredderivative) plasma exposure curve (i.e., concentration in plasma in pMvs time) from time 30 to 180 min is determined (i.e., the result may beindicated in (min x pM/pmol) or simply in min/L).298. The derivative of embodiment 297, wherein the AUC of thedose-corrected plasma exposure curve isa) at least 10, preferably at least 20, or more preferably at least 30min/L;b) at least 40, preferably at least 50, more preferably at least 60, ormost preferably at least 70 min/L; orc) at least 1.5 times, preferably at least 2 times, more preferably atleast 3 times, or most preferably at least 4 times the corresponding AUCvalue for semaglutide.299. The derivative of any of embodiments 294-298, wherein the GLP-1derivative is tested in a concentration of about 1000 uM in a solutionof 250 mg/ml of sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC).300. The derivative of any of embodiments 294-299, wherein male SpragueDawley rats are used, preferably with a body weight upon arrival ofapproximately 240 g.301. The derivative of any of embodiments 294-300, wherein the rats arefasted for approximately 18 hours before the experiment.302. The derivative of any of embodiments 294-301, wherein the rats areand taken into general anaesthesia after having fasted and before theinjection of the derivative in the jejunum, or the oral gavage,respectively.303. The derivative of any of embodiments 294-302, wherein for injectionin the intestinal lumen the derivative is administered in the proximalpart of the jejunum (10 cm distal for the duodenum) or in themid-intestine (50 cm proximal for the cecum), preferably in the proximalpart of the jejunum.304. The derivative of any of embodiments 294-303, wherein 100 μl of thederivative is injected into the jejunal lumen through a catheter with a1 ml syringe, and subsequently 200 μl of air is pushed into the jejunallumen with another syringe, which is then left connected to the catheterto prevent flow back into the catheter.305. The derivative of any of embodiments 294-304, wherein blood samples(200 ul) are collected into EDTA tubes from the tail vein at desiredintervals, such as at times 0, 10, 30, 60, 120 and 240 min, andcentrifuged 5 minutes, 10000 G, at 4° C. within 20 minutes.306. The derivative of any of embodiments 294-305, wherein plasma (e.g.75 ul) is separated, immediately frozen, and kept at −20° C. untilanalyzed for plasma concentration of the derivative.307. The derivative of any of embodiments 294-306, wherein LOCI(Luminescent Oxygen Channeling Immunoassay) is used for analyzing theplasma concentration of the derivative.308. An intermediate product in the form of a GLP-1 analogue whichcomprises the following change as compared to GLP-1(7-37) (SEQ ID NO:1): (i) 38Q; and/or (ii) 39G; or a pharmaceutically acceptable salt,amide, or ester thereof.309. The GLP-1 analogue of embodiment 308 which comprises (38E, 39G).310. An intermediate product in the form of a GLP-1 analogue whichcomprises, the following amino acid changes, as compared to GLP-1(7-37)(SEQ ID NO: 1): (i) 22E, 26R, 27K, 34R, 37K; (ii) 22E, 26R, 27K, 30E,34R, 36K, 38E, 39G; (iii) 22E, 26R, 27K, 34R, 36K, des37; (iv) 22E, 25V,26R, 27K, 34R, 37K; (v) 8Aib, 20K, 22E, 26R, 27K, 30E, 34G, des35-37;(vi) 26R, 27K, 30E, 34R, 36K, 38E; (vii) 8Aib, 22K, 25V, 26R, 27K, 31H,34R; (iix) 8Aib, 22K, 25V, 26R, 27K, 34R, des35-37; (ix) 8Aib, 22K, 25V,26R, 27K, 34R, des36-37; (x) 26H, 27K, 30E, 34R, 36K, 38E; (xi) 22K,25V, 26R, 27K, 30E, 34Q; (xii) 25V, 26R, 27K, 30E, 34R, 36K, 38Q; (xiii)25V, 26R, 27K, 30E, 34Q, 36K, 38E; (xiv) 22K, 26R, 27K, 31H, 34G,des35-37; (xv) 8Aib, 25V, 26R, 27K, 31H, 34Q, 37K; (xvi) 25V, 26R, 27K,31H, 34Q, 37K; (xvii) 22E, 23E, 25V, 26R, 27K, 31H, 34Q, 37K; (iixx)8Aib, 12K, 22E, 26R, 27K, 31H, 34Q; (ixx) 8Aib, 22K, 26R, 27K, 31H, 34G,des35-37; (xx) 22E, 26H, 27K, 30E, 34R, 36K, 38E; (xxi) 22E, 24K, 26R,27K, 31H, 34G, des35-37; (xxii) 25V, 26R, 27K, 34Q, 36K; (xxiii) 22E,24K, 25V, 26R, 27K, 31H, 34R; (xxiv) 22E, 24K, 25V, 26R, 27K, 34G,des35-37; (xxv) 22E, 24K, 25V, 26R, 27K, 34R; (xxvi) 8Aib, 22E, 24K,25V, 26R, 27K, 31H, 34Q; or (xxvii) 8Aib, 22E, 26R, 27K, 30E, 34R, 36K,38E, 39G; or a pharmaceutically acceptable salt, amide, or esterthereof.311. The GLP-analogue of embodiment 310 which has a set of amino acidchanges as defined in any of (i)-(xxvii).312. A derivative according to any of embodiments 1-307, for use as amedicament.313. A derivative according to any of embodiments 1-307, for use in thetreatment and/or prevention of all forms of diabetes and relateddiseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression.314. A method for treating or preventing all forms of diabetes andrelated diseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression—by administering a pharmaceutically active amount ofa derivative according to any of embodiments 1-307.

The following are additional particular embodiments of the invention:

1. A derivative of a GLP-1 analogue,

which analogue comprises a first K residue at a position correspondingto position 27 of GLP-1(7-37) (SEQ ID NO: 1); a second K residue at aposition corresponding to position T of GLP-1(7-37), where T is aninteger in the range of 7-37 except 18 and 27; and a maximum of tenamino acid changes as compared to GLP-1(7-37); wherein the first Kresidue is designated K²⁷, and the second K residue is designated K^(T);

-   -   which derivative comprises two albumin binding moieties attached        to K²⁷ and K^(T), respectively, wherein

the albumin binding moiety comprises a protracting moiety selected fromChem. 1 and Chem. 2:

HOOC—(CH₂)_(x)—CO—*  Chem. 1:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

in which x is an integer in the range of 6-18, and y is an integer inthe range of 3-17;

with the proviso that when the protracting moiety is Chem. 1, thealbumin binding moiety further comprises a linker of formula Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; or a pharmaceutically acceptable salt, amide, or esterthereof.

2. The derivative of embodiment 1,

wherein the GLP-1 analogue comprises a first K residue at a positioncorresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1); a second Kresidue at a position corresponding to position T of GLP-1(7-37), whereT is an integer in the range of 7-37 except 18 and 27; and a maximum often amino acid changes as compared to GLP-1(7-37); wherein the first Kresidue is designated K²⁷, and the second K residue is designated K^(T);

which derivative comprises two albumin binding moieties attached to K²⁷and K^(T), respectively, wherein

the albumin binding moiety comprises a protracting moiety of Chem. 2:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

in which y is an integer in the range of 3-17;

or a pharmaceutically acceptable salt, amide, or ester thereof.

3. The derivative of any of the previous embodiments, wherein thealbumin binding moiety further comprises a linker.4. The derivative of any of the previous embodiments, wherein the linkercomprises i) a Glu di-radical; and/or ii) a linker of formula Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5.5. The derivative of any of the previous embodiments, wherein the Gludi-radical is selected from Chem. 6, and/or Chem. 7:

preferably Chem. 6.6. The derivative of any of the previous embodiments,

wherein the GLP-1 analogue comprises a first K residue at a positioncorresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1); a second Kresidue at a position corresponding to position T of GLP-1(7-37), whereT is an integer in the range of 7-37 except 18 and 27; and a maximum often amino acid changes as compared to GLP-1(7-37); wherein the first Kresidue is designated K²⁷, and the second K residue is designated K^(T);

which derivative comprises two albumin binding moieties attached to K²⁷and K^(T), respectively, wherein

the albumin binding moiety comprises

i) a protracting moiety of formula Chem. 1:

HOOC—(CH₂)_(x)—CO—*  Chem. 1:

in which x is an integer in the range of 6-18; and

ii) a linker of formula Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; or a pharmaceutically acceptable salt, amide, or esterthereof.

7. The derivative of any of the previous embodiments,

wherein the GLP-1 analogue comprises a first K residue at a positioncorresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1); a second Kresidue at a position corresponding to position T of GLP-1(7-37), whereT is an integer in the range of 7-37 except 18 and 27; and a maximum often amino acid changes as compared to GLP-1(7-37); wherein the first Kresidue is designated K²⁷, and the second K residue is designated K^(T);

which derivative comprises two protracting moieties attached to K²⁷ andK^(T), respectively, via a linker, wherein

the protracting moiety is selected from Chem. 1 and Chem. 2:

HOOC—(CH₂)_(x)—CO—*  Chem. 1:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

in which x is an integer in the range of 6-18, y is an integer in therange of 3-17; and

the linker comprises Chem. 5: Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5;

or a pharmaceutically acceptable salt, amide, or ester thereof.

8. The derivative of any of the previous embodiments, wherein T is aninteger selected from the range of 7-37 except 18 and 27.9. The derivative of any of the previous embodiments, wherein T isselected from any of the ranges of 7-17, 19-26, and 28-37.10. The derivative of any of the previous embodiments, wherein T isselected from the range of 7-17.11. The derivative of any of the previous embodiments, wherein T is 12.12. The derivative of any of the previous embodiments, wherein T isselected from the range of 19-26.13. The derivative of any of the previous embodiments, wherein T isselected from the group consisting of 20, 22, and 24.14. The derivative of any of the previous embodiments, wherein T is 20.15. The derivative of any of the previous embodiments, wherein T is 22or 24.16. The derivative of any of the previous embodiments, wherein T is 22.17. The derivative of any of the previous embodiments, wherein T is 24.18. The derivative of any of the previous embodiments, wherein T isselected from the range of 28-37.19. The derivative of any of the previous embodiments, wherein T isselected from the group consisting of 36 and 37.20. The derivative of any of the previous embodiments, wherein T is 36.21. The derivative of any of the previous embodiments, wherein T is 37.22. The derivative of any of the previous embodiments, wherein theposition corresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1) isidentified by handwriting and eyeballing.23. The derivative of any of the previous embodiments, wherein theposition corresponding to position T of GLP-1(7-37) (SEQ ID NO: 1) isidentified by handwriting and eyeballing.24. The derivative of any of the previous embodiments, wherein theposition corresponding to position 27 of GLP-1(7-37) (SEQ ID NO: 1) isidentified by use of a standard protein or peptide alignment program.25. The derivative of any of the previous embodiments, wherein theposition corresponding to position T of GLP-1(7-37) (SEQ ID NO: 1) isidentified by use of a standard protein or peptide alignment program.26. The derivative of any of the previous embodiments, wherein thealignment program is a Needleman-Wunsch alignment.27. The derivative of any of the previous embodiments, wherein thedefault scoring matrix and the default identity matrix is used.28. The derivative of any of the previous embodiments, wherein thescoring matrix is BLOSUM62.29. The derivative of any of the previous embodiments, wherein thepenalty for the first residue in a gap is −10 (minus ten).30. The derivative of any of the previous embodiments, wherein thepenalties for additional residues in a gap is −0.5 (minus point five).31. The derivative of any of the previous embodiments, wherein theanalogue comprises no K residues other than the first and the second Kresidue.32. The derivative of any of the previous embodiments, wherein theprotracting moiety is Chem. 1.33. The derivative of any of the previous embodiments, wherein x is aneven number.34. The derivative of any of the previous embodiments, wherein x is 12.35. The derivative of any of the previous embodiments, wherein Chem. 1is represented by Chem. 1a:

where x is as defined in any of the previous embodiments.36. The derivative of any of the previous embodiments, wherein theprotracting moiety is Chem. 2, preferably Chem. 2a:

wherein y is as defined in any of the previous embodiments.37. The derivative of any of the previous embodiments, wherein y is anodd number.38. The derivative of any of the previous embodiments, wherein y is 9.39. The derivative of any of the previous embodiments, wherein Chem. 2is represented by Chem. 2b, or Chem. 2c:

preferably by Chem. 2b;wherein y is as defined in any of the previous embodiments.39a. The derivative of any of the previous embodiments, wherein Chem. 2ais represented by Chem. 2b, or Chem. 2c:

preferably by Chem. 2b;wherein y is as defined in any of the previous embodiments.40. The derivative of any of the previous embodiments, which comprisesChem. 5.41. The derivative of any of the previous embodiments, wherein Chem. 5is a first linker element.42. The derivative of any of the previous embodiments, wherein k is 1.43. The derivative of any of the previous embodiments, wherein n is 1.44. The derivative of any of the previous embodiments, wherein Chem. 5is included m times, wherein m is an integer in the range of 1-10.45. The derivative of any of the previous embodiments, wherein m is 2.46. The derivative of any of the previous embodiments, wherein, when mis not 1, the Chem. 5 elements are interconnected via amide bond(s).47. The derivative of any of the previous embodiments, wherein thelinker further comprises a second linker element; preferably a Gludi-radical; more preferably selected from Chem. 6, and/or Chem. 7:

most preferably Chem. 6.48. The derivative of any of the previous embodiments, wherein the Gludi-radical is included p times, wherein p is an integer in the range of1-2.49. The derivative of any of the previous embodiments, wherein p is 1.50. The derivative of any of the previous embodiments, wherein p is 2.51. The derivative of any of the previous embodiments, wherein the Gludi-radical is a radical of L-Glu.52. The derivative of any of the previous embodiments, wherein the oneor more Glu di-radicals and the one or more Chem. 5 elements areinterconnected via amide bond(s).53. The derivative of any of the previous embodiments, wherein thelinker consists of m times Chem. 5 and p times the Glu di-radical.54. The derivative of any of the previous embodiments, wherein (m,p) is(2,2) or (2,1), preferably (2,1).55. The derivative of the previous embodiments, wherein the m Chem. 5elements and the p Glu di-radicals are interconnected via amide bonds.56. The derivative of any of the previous embodiments, wherein thelinker and the protracting moiety are interconnected via an amide bond.57. The derivative of any of the previous embodiments, wherein thelinker and the GLP-1 analogue are interconnected via an amide bond.58. The derivative of any of the previous embodiments, wherein thelinker is attached to the epsilon-amino group of the first or the secondK residue.59. The derivative of any of the previous embodiments, wherein thelinker has from 5 to 41 C-atoms; preferably 17 or 22 C-atoms.60. The derivative of any of the previous embodiments, wherein thelinker has 17 C-atoms.61. The derivative of any of the previous embodiments, wherein thelinker has 22 C-atoms.62. The derivative of the previous embodiments, wherein the linker hasfrom 4 to 28 hetero atoms; preferably 12 or 16 hetero atoms.63. The derivative of any of the previous embodiments, wherein thelinker has 12 hetero atoms.64. The derivative of any of the previous embodiments, wherein thelinker has 16 hetero atoms.65. The derivative of any of the previous embodiments, wherein thehetero atoms are N-, and/or O-atoms.66. The derivative of any of the previous embodiments, wherein thelinker has from 1 to 7 N-atoms; preferably 3 or 4 N-atoms.67. The derivative of any of the previous embodiments, wherein thelinker has 3 N-atoms.68. The derivative of any of the previous embodiments, wherein thelinker has 4 N-atoms.69. The derivative of any of the previous embodiments, wherein thelinker has from 3 to 21 O-atoms; preferably 9 or 12 O-atoms.70. The derivative of any of the previous embodiments, wherein thelinker has 9 O-atoms.71. The derivative of any of the previous embodiments, wherein thelinker has 12 O-atoms.72. The derivative of any of the previous embodiments, wherein thelinker consists of two times Chem. 6 and two times Chem. 5,interconnected via amide bonds and in the sequence indicated, the linkerbeing connected at its *—NH end to the *—CO end of the protractingmoiety, and at its *—CO end to the epsilon amino group of K²⁷ or K^(T)of the GLP-1 analogue.73. The derivative of any of the previous embodiments, wherein thelinker consists of two times Chem. 5 and one time Chem. 6,interconnected via amide bonds and in the sequence indicated, the linkerbeing connected at its *—NH end to the *—CO end of the protractingmoiety, and at its free *—CO end to the epsilon amino group of K²⁷ orK^(T) of the GLP-1 analogue.74. The derivative of any of the previous embodiments, wherein thelinker consists of one time Chem. 6 and two times Chem. 5,interconnected via amide bonds and in the sequence indicated, the linkerbeing connected at its *—NH end to the *—CO end of the protractingmoiety, and at its *—CO end to the epsilon amino group of K²⁷ or K^(T)of the GLP-1 analogue.75. The derivative of any of the previous embodiments, wherein thelinker consists of one time Chem. 6, two times Chem. 5, and one timeChem. 6, interconnected via amide bonds and in the sequence indicated,the linker being connected at its *—NH end to the *—CO end of theprotracting moiety, and at its *—CO end to the epsilon amino group ofK²⁷ or K^(T) of the GLP-1 analogue.76. The derivative of any of the previous embodiments, wherein the twoprotracting moieties are substantially identical; such as at least 80%,at least 85%, at least 90%, at least 95%, or at least 99% identical.77. The derivative of any of the previous embodiments, wherein the twoprotracting moieties have a similarity of at least 0.5; preferably atleast 0.6; more preferably at least 0.7, or at least 0.8; even morepreferably at least 0.9; or most preferably at least 0.99, such as asimilarity of 1.0.78. The derivative of any of the previous embodiments, wherein the twolinkers have a similarity of at least 0.5; preferably at least 0.6; morepreferably at least 0.7, or at least 0.8; even more preferably at least0.9; or most preferably at least 0.99, such as a similarity of 1.0.79. The derivative of any of the previous embodiments, wherein the twoalbumin binders, such as the two side chains consisting of protractingmoiety and linker, are substantially identical; such as at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% identical.80. The derivative of any of the previous embodiments, wherein the twoalbumin binders, such as the two side chains consisting of protractingmoiety and linker, have a similarity of at least 0.5; preferably atleast 0.6; more preferably at least 0.7, or at least 0.8; even morepreferably at least 0.9; or most preferably at least 0.99, such as asimilarity of 1.0.81. The derivative of any of the previous embodiments, wherein the twochemical structures to be compared are represented as fingerprints, suchas a) ECFP_6 fingerprints; b) UNITY fingerprints; and/or c) MDLfingerprints; and wherein for each of a), b) and c) the Tanimotocoefficient is preferably used for calculating the similarity, oridentity, of the two fingerprints.82. The derivative of any of the previous embodiments, wherein thenumber of amino acid changes as compared to GLP-1(7-37) (SEQ ID NO: 1)are identified by handwriting and eyeballing.83. The derivative of any of the previous embodiments, wherein thenumber of amino acid changes as compared to GLP-1(7-37) (SEQ ID NO: 1)are identified by use of a standard protein or peptide alignmentprogram.84. The derivative of any of the previous embodiments, wherein thealignment program is a Needleman-Wunsch alignment.85. The derivative of any of the previous embodiments, wherein thedefault scoring matrix and the default identity matrix is used.86. The derivative of any of the previous embodiments, wherein thescoring matrix is BLOSUM62.87. The derivative of any of the previous embodiments, wherein thepenalty for the first residue in a gap is −10 (minus ten).88. The derivative of any of the previous embodiments, wherein thepenalties for additional residues in a gap is −0.5 (minus point five).89. The derivative of any of the previous embodiments, wherein the aminoacid change(s) is (are) at one or more positions corresponding to thefollowing positions in GLP-1(7-37) (SEQ ID NO: 1): 8, 12, 20, 22, 23,24, 25, 26, 27, 30, 31, 34, 35, 36, 37, 38, and 39.90. The derivative of any of the previous embodiments, wherein theanalogue comprises at least one of the following changes: Aib⁸, K¹²,K²⁰, E²² or K²², E²³, K²⁴, V²⁵, R²⁶ or H²⁶, K²⁷, E³⁰, H³¹, G³⁴ or R³⁴ orQ³⁴, Des³⁵, K³⁶ or Des³⁶, K³⁷ or Des³⁷, E³⁸ or Q³⁸, and/or G³⁹.91. The derivative of any of the previous embodiments, wherein thesecond K residue is K¹², and wherein the analogue, in addition to thechange K²⁷, further comprises i) a change selected from G³⁴ and Q³⁴, andii) a change selected from R²⁶ and H²⁶.92. The derivative of any of the previous embodiments, wherein thesecond K residue is K²⁰, and wherein the analogue, in addition to thechange K²⁷, further comprises i) a change selected from G³⁴ and Q³⁴, andii) a change selected from R²⁶ and H²⁶.93. The derivative of any of the previous embodiments, wherein thesecond K residue is K²², and wherein the analogue, in addition to thechange K²⁷, further comprises i) a change selected from G³⁴ and Q³⁴, andii) a change selected from R²⁶ and H²⁶.94. The derivative of any of the previous embodiments, wherein thesecond K residue is K²⁴, and wherein the analogue, in addition to thechange K²⁷, further comprises i) a change selected from G³⁴ and Q³⁴, andii) a change selected from R²⁶ and H²⁶.95. The derivative of any of the previous embodiments, wherein thesecond K residue is K³⁶, and wherein the analogue, in addition to thechange K²⁷, further comprises i) a change selected from G³⁴ and Q³⁴, andii) a change selected from R²⁶ and H²⁶.96. The derivative of any of the previous embodiments, wherein thesecond K residue is K³⁷, and wherein the analogue, in addition to thechange K²⁷, further comprises i) a change selected from G³⁴ and Q³⁴, andii) a change selected from R²⁶ and H²⁶.97. The derivative of any of the previous embodiments, wherein theanalogue comprises at least one of the following changes: Aib⁸, E²²,E²³, V²⁵, E³⁰, H³¹, Des³⁵, Des³⁶, Des³⁷, E³⁸ or Q³⁸, and/or G³⁹.98. The derivative of any of the previous embodiments, wherein theanalogue comprises Aib⁸.99. The derivative of any of the previous embodiments, wherein theanalogue comprises E²².100. The derivative of any of the previous embodiments, wherein theanalogue comprises E²³.101. The derivative of any of the previous embodiments, wherein theanalogue comprises V²⁵.102. The derivative of any of the previous embodiments, wherein theanalogue comprises E³⁰.103. The derivative of any of the previous embodiments, wherein theanalogue comprises H³¹.104. The derivative of any of the previous embodiments, wherein theanalogue comprises Des³⁵.105. The derivative of any of the previous embodiments, wherein theanalogue comprises Des³⁶.106. The derivative of any of the previous embodiments, wherein theanalogue comprises Des³⁷.107. The derivative of any of the previous embodiments, wherein theanalogue comprises E³⁸ or Q³⁸, preferably Q³⁸, or more preferably E³⁸.108. The derivative of any of the previous embodiments, wherein theanalogue comprises G³⁹.109. The derivative of any of the previous embodiments, wherein theanalogue comprises Des³⁵, Des³⁶, and Des³⁷.110. The derivative of any of the previous embodiments, wherein theanalogue comprises Des³⁶ and Des³⁷.111. The derivative of any of the previous embodiments, which is aderivative of GLP-1(7-34) (amino acids 1-28 of SEQ ID NO: 1).112. The derivative of any of the previous embodiments, which is aderivative of GLP-1(7-35) (amino acids 1-29 of SEQ ID NO: 1).113. The derivative of any of the previous embodiments, which is aderivative of GLP-1(7-36) (amino acids 1-30 of SEQ ID NO: 1).114. The derivative of any of the previous embodiments, which is aderivative of GLP-1(7-37) (amino acids 1-31 of SEQ ID NO: 1).115. The derivative of any of the previous embodiments, which is aderivative of GLP-1(7-38) (amino acids 1-31 of SEQ ID NO: 1, plus oneC-terminally added amino acid residue).116. The derivative of any of the previous embodiments, which is aderivative of GLP-1(7-39) (amino acids 1-31 of SEQ ID NO: 1, plus twoC-terminally added amino acid residues).117. The derivative of any of the previous embodiments, wherein theanalogue has a maximum of ten amino acid changes.118. The derivative of any of the previous embodiments, wherein theanalogue has a maximum of nine amino acid changes.119. The derivative of any of the previous embodiments, wherein theanalogue has a maximum of eight amino acid changes.120. The derivative of any of the previous embodiments, wherein theanalogue has a maximum of seven amino acid changes.121. The derivative of any of the previous embodiments, wherein theanalogue has a maximum of six amino acid changes.122. The derivative of any of the previous embodiments, wherein theanalogue has a maximum of five amino acid changes.123. The derivative of any of the previous embodiments, wherein theanalogue has a maximum of four amino acid changes.124. The derivative of any of the previous embodiments, wherein theanalogue has a maximum of three amino acid changes.125. The derivative of any of the previous embodiments, wherein theanalogue has a maximum of two amino acid changes.126. The derivative of any of the previous embodiments, wherein theanalogue has a maximum of one amino acid changes.127. The derivative of any of the previous embodiments, wherein theanalogue has a minimum of one amino acid changes.128. The derivative of any of the previous embodiments, wherein theanalogue has a minimum of two amino acid changes.129. The derivative of any of the previous embodiments, wherein theanalogue has a minimum of three amino acid changes.130. The derivative of any of the previous embodiments, wherein theanalogue has a minimum of four amino acid changes.131. The derivative of any of the previous embodiments, wherein theanalogue has a minimum of five amino acid changes.132. The derivative of any of the previous embodiments, wherein theanalogue has a minimum of six amino acid changes.133. The derivative of any of the previous embodiments, wherein theanalogue has a minimum of seven amino acid changes.134. The derivative of any of the previous embodiments, wherein theanalogue has a minimum of eight amino acid changes.135. The derivative of any of the previous embodiments, wherein theanalogue has a minimum of nine amino acid changes.136. The derivative of any of the previous embodiments, wherein theanalogue has a minimum of ten amino acid changes.137. The derivative of any of the previous embodiments, wherein theanalogue has one amino acid change.138. The derivative of any of the previous embodiments, wherein theanalogue has two amino acid changes.139. The derivative of any of the previous embodiments, wherein theanalogue has three amino acid changes.140. The derivative of any of the previous embodiments, wherein theanalogue has four amino acid changes.141. The derivative of any of the previous embodiments, wherein theanalogue has five amino acid changes.142. The derivative of any of the previous embodiments, wherein theanalogue has six amino acid changes.143. The derivative of any of the previous embodiments, wherein theanalogue has seven amino acid changes.144. The derivative of any of the previous embodiments, wherein theanalogue has eight amino acid changes.145. The derivative of any of the previous embodiments, wherein theanalogue has nine amino acid changes.146. The derivative of any of the previous embodiments, wherein theanalogue has ten amino acid changes.147. The derivative of any of the previous embodiments, wherein thechange(s) is (are), independently, substitutions, additions, and/ordeletions.148. The derivative of any of the previous embodiments, wherein theanaloguea) comprises a GLP-1 analogue of Formula I (SEQ ID NO: 3); and/or b) isa GLP-1 analogue of Formula I (SEQ ID NO: 3):

Xaa₇-Xaa₈-Glu-Gly-Thr-Xaa₁₂-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Xaa₂₄-Xaa₂₅-Xaa₂₆-Lys-Phe-Ile-Xaa₃₀-Xaa₃₁-Leu-Val-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉,wherein  Formula I:

Xaa₇ is L-histidine, imidazopropionyl, α-hydroxy-histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, N^(α)-acetyl-histidine, N^(α)-formyl-histidine,α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine,2-pyridylalanine, or 4-pyridylalanine;

Xaa₈ is Ala, Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or(1-aminocyclooctyl) carboxylic acid;

Xaa₁₂ is Lys or Phe;

Xaa₁₆ is Val or Leu;

Xaa₁₈ is Ser, Arg, Asn, Gin, or Glu;

Xaa₁₉ is Tyr or Gin;

Xaa₂₀ is Leu, Lys, or Met;

Xaa₂₂ is Gly, Glu, Lys, or Aib;

Xaa₂₃ is Gin, Glu, or Arg;

Xaa₂₄ is Ala or Lys;

Xaa₂₅ is Ala or Val;

Xaa₂₆ is Val, His, or Arg;

Xaa₃₀ is Ala, Glu, or Arg;

Xaa₃₁ is Trp or His;

Xaa₃₄ is Glu, Asn, Gly, Gin, or Arg;

Xaa₃₅ is Gly, Aib, or absent;

Xaa₃₆ is Arg, Gly, Lys, or absent;

Xaa₃₇ is Gly, Ala, Glu, Pro, Lys, Arg, or absent;

Xaa₃₈ is Ser, Gly, Ala, Glu, Gin, Pro, Arg, or absent; and

Xaa₃₉ is Gly or absent.

149. The derivative of any of the previous embodiments wherein thepeptide of Formula I (SEQ ID NO: 3) is an analogue of GLP-1(7-37) (SEQID NO: 1).150. The derivative of any of the previous embodiments, wherein if Xaa₃₈is absent, then Xaa₃₉ is also absent.151. The derivative of any of the previous embodiments, wherein if Xaa₃₇is absent, then Xaa₃₈ and Xaa₃₉ are also absent.152. The derivative of any of the previous embodiments, wherein if Xaa₃₆is absent, then Xaa₃₇, Xaa₃₈, and Xaa₃₉ are also absent.153. The derivative of any of the previous embodiments, wherein if Xaa₃₅is absent, then Xaa₃₆, Xaa₃₇, Xaa₃₈, and Xaa₃₉ are also absent.154. The derivative of any of the previous embodiments, wherein Xaa₇ isHis; Xaa₈ is Ala or Aib; Xaa₁₂ is Lys or Phe; Xaa₁₆ is Val; Xaa₁₈ isSer; Xaa₁₉ is Tyr; Xaa₂₀ is Leu or Lys; Xaa₂₂ is Glu, Gly or Lys; Xaa₂₃is Gin or Glu; Xaa₂₄ is Ala or Lys; Xaa₂₅ is Ala or Val; Xaa₂₆ is His orArg; Xaa₃₀ is Ala or Glu; Xaa₃₁ is Trp or His; Xaa₃₄ is Gly, Gin, orArg; Xaa₃₅ is Gly or absent; Xaa₃₆ is Arg, Lys, or absent; Xaa₃₇ is Gly,Lys, or absent; Xaa₃₈ is Glu or Gin; and Xaa₃₉ is Gly or absent.154a. The derivative of any of the previous embodiments, wherein Xaa₇ isHis.154b. The derivative of any of the previous embodiments, wherein Xaa₈ isAla.154b1. The derivative of any of the previous embodiments, wherein Xaa₈is Aib.154c. The derivative of any of the previous embodiments, wherein Xaa₁₂is Lys.154d. The derivative of any of the previous embodiments, wherein Xaa₁₂is Phe.154e. The derivative of any of the previous embodiments, wherein Xaa₁₆is Val.154f. The derivative of any of the previous embodiments, wherein Xaa₁₈is Ser.154 g. The derivative of any of the previous embodiments, wherein Xaa₁₉is Tyr.154 h. The derivative of any of the previous embodiments, wherein Xaa₂₀is Leu.154i. The derivative of any of the previous embodiments, wherein Xaa₂₀is Lys.154j. The derivative of any of the previous embodiments, wherein Xaa₂₂is Glu.154k. The derivative of any of the previous embodiments, wherein Xaa₂₂is Gly.154l. The derivative of any of the previous embodiments, wherein Xaa₂₂is Lys.154m. The derivative of any of the previous embodiments, wherein Xaa₂₃is Gin.154n. The derivative of any of the previous embodiments, wherein Xaa₂₃is Glu.1540. The derivative of any of the previous embodiments, wherein Xaa₂₄is Ala.154p. The derivative of any of the previous embodiments, wherein Xaa₂₄is Lys.154q. The derivative of any of the previous embodiments, wherein Xaa₂₅is Ala.154r. The derivative of any of the previous embodiments, wherein Xaa₂₅is Val.154s. The derivative of any of the previous embodiments, wherein Xaa₂₆is His.154t. The derivative of any of the previous embodiments, wherein Xaa₂₆is Arg.154u. The derivative of any of the previous embodiments, wherein Xaa₃₀is Ala.154v. The derivative of any of the previous embodiments, wherein Xaa₃₀is Glu.154x. The derivative of any of the previous embodiments, wherein Xaa₃₁is Trp.154y. The derivative of any of the previous embodiments, wherein Xaa₃₁is His.154z. The derivative of any of the previous embodiments, wherein Xaa₃₄is Gly.154aa. The derivative of any of the previous embodiments, wherein Xaa₃₄is Gin.154ab. The derivative of any of the previous embodiments, wherein Xaa₃₄is Arg.154ac. The derivative of any of the previous embodiments, wherein Xaa₃₅is Gly.154ad. The derivative of any of the previous embodiments, wherein Xaa₃₅is absent.154ae. The derivative of any of the previous embodiments, wherein Xaa₃₆is Arg.154af. The derivative of any of the previous embodiments, wherein Xaa₃₆is Lys.154ag. The derivative of any of the previous embodiments, wherein Xaa₃₆is absent.154ah. The derivative of any of the previous embodiments, wherein Xaa₃₇is Gly.154ai. The derivative of any of the previous embodiments, wherein Xaa₃₇is Lys.154aj. The derivative of any of the previous embodiments, wherein Xaa₃₇is absent.154ak. The derivative of any of the previous embodiments, wherein Xaa₃₈is Glu.154al. The derivative of any of the previous embodiments, wherein Xaa₃₈is Gin.154am. The derivative of any of the previous embodiments, wherein Xaa₃₈is absent.154an. The derivative of any of the previous embodiments, wherein Xaa₃₉is Gly.154ao. The derivative of any of the previous embodiments, wherein Xaa₃₉is absent.155. The derivative of any of the previous embodiments, wherein theanalogue comprises, preferably has, the following amino acid changes, ascompared to GLP-1(7-37) (SEQ ID NO: 1): (i) 22E, 26R, 27K, 34R, 37K;(ii) 22E, 26R, 27K, 30E, 34R, 36K, 38E, 39G; (iii) 22E, 26R, 27K, 34R,36K, des37; (iv) 22E, 25V, 26R, 27K, 34R, 37K; (v) 8Aib, 20K, 22E, 26R,27K, 30E, 34G, des35-37; (vi) 26R, 27K, 30E, 34R, 36K, 38E; (vii) 8Aib,22K, 25V, 26R, 27K, 31H, 34R; (iix) 8Aib, 22K, 25V, 26R, 27K, 34R,des35-37; (ix) 8Aib, 22K, 25V, 26R, 27K, 34R, des36-37; (x) 26H, 27K,30E, 34R, 36K, 38E; (xi) 22K, 25V, 26R, 27K, 30E, 34Q; (xii) 25V, 26R,27K, 30E, 34R, 36K, 38Q; (xiii) 25V, 26R, 27K, 30E, 34Q, 36K, 38E; (xiv)22K, 26R, 27K, 31H, 34G, des35-37; (xv) 8Aib, 25V, 26R, 27K, 31H, 34Q,37K; (xvi) 25V, 26R, 27K, 31H, 34Q, 37K; (xvii) 22E, 23E, 25V, 26R, 27K,31H, 34Q, 37K; (iixx) 8Aib, 12K, 22E, 26R, 27K, 31H, 34Q; (ixx) 8Aib,22K, 26R, 27K, 31H, 34G, des35-37; (xx) 22E, 26H, 27K, 30E, 34R, 36K,38E; (xxi) 22E, 24K, 26R, 27K, 31H, 34G, des35-37; (xxii) 25V, 26R, 27K,34Q, 36K; (xxiii) 22E, 24K, 25V, 26R, 27K, 31H, 34R; (xxiv) 22E, 24K,25V, 26R, 27K, 34G, des35-37; (xxv) 22E, 24K, 25V, 26R, 27K, 34R; or(xxvi) 8Aib, 22E, 24K, 25V, 26R, 27K, 31H, 34Q.156. A compound, preferably according to any of the previousembodiments, selected from the following: Chem. 50, Chem. 51, Chem. 52,Chem. 53, Chem. 54, Chem. 55, Chem. 56, Chem. 57, Chem. 58, Chem. 59,Chem. 60, Chem. 61, Chem. 62, Chem. 63, Chem. 64, Chem. 65, Chem. 66,Chem. 67, Chem. 68, Chem. 69, Chem. 70, Chem. 71, Chem. 72, Chem. 73,Chem. 74, Chem. 75, Chem. 76, Chem. 77, Chem. 78, and Chem. 79; or apharmaceutically acceptable salt, amide, or ester thereof.157. A compound, preferably according to any of the previousembodiments, characterised by its name, and selected from a listing ofeach of the names of the compounds of Examples 1-30 herein, or apharmaceutically acceptable salt, amide, or ester thereof.158. The derivative of any of the previous embodiments, which has GLP-1activity.159. The derivative of any of the previous embodiments, wherein GLP-1activity refers to the capability of activating the human GLP-1receptor.160. The derivative of any of the previous embodiments, whereinactivation of the human GLP-1 receptor is measured in an in vitro assay,as the potency of cAMP production.161. The derivative of any of the previous embodiments, which has apotency corresponding to an EC₅₀a) below 10000 pM, more preferably below 5000 pM, even more preferablybelow 4000 pM, or most preferably below 3000 pM;b) below 2000 pM, preferably below 1500 pM, more preferably below 1200pM, even more preferably below 1000 pM, or most preferably below 500 pM;c) below 400 pM, preferably below 300 pM, more preferably below 200 pM,even more preferably below 150 pM, or most preferably below 100 pM; ord) below 80 pM, preferably below 60 pM, more preferably below 40 pM,even more preferably below 30 pM, or most preferably below 20 pM.162. The derivative of any of the previous embodiments, wherein thepotency is determined as EC₅₀ for the dose-response curve showingdose-dependent formation of cAMP in a medium containing the human GLP-1receptor, preferably using a stable transfected cell-line such asBHK467-12A (tk-ts13), and/or using for the determination of cAMP afunctional receptor assay, e.g. based on competition betweenendogenously formed cAMP and exogenously added biotin-labelled cAMP, inwhich assay cAMP is more preferably captured using a specific antibody,and/or wherein an even more preferred assay is the AlphaScreen cAMPAssay, most preferably the one described in Example 31.163. The derivative of any of the previous embodiments, for which theratio [GLP-1 receptor binding affinity (IC₅₀) in the presence of 2.0%HSA (high albumin), divided by GLP-1 receptor binding affinity (IC₅₀) inthe presence of 0.005% HSA (low albumin)] is:a) at least 1.0, more preferably at least 10, even more preferably atleast 25, or most preferably at least 50;b) at least 60, preferably at least 70, more preferably at least 80,even more preferably at least 90, or most preferably at least 100;c) at least 125, preferably at least 150, more preferably at least 200,still more preferably at least 250, even more preferably at least 400,or most preferably at least 500; ord) at least 600, preferably at least 800, even more preferably at least900, or most preferably at least 1000.164. The derivative of any of the previous embodiments, for which theGLP-1 receptor binding affinity (IC₅₀) in the presence of 0.005% HSA(low albumin) isa) below 1000 nM, preferably below 750 nM, more preferably below 500 nM,or most preferably below 100 nM; orb) below 50.0 nM, preferably below 15.0 nM, more preferably below 10.0nM, even more preferably below 5.0 nM, or most preferably below 1.0 nM.165. The derivative of any of the previous embodiments, for which theGLP-1 receptor binding affinity (IC₅₀) in the presence of 2.0% HSA (highalbumin) isa) below 1100 nM, preferably below 1000 nM, more preferably below 900nM, or most preferably below 600 nM; orb) below 500 nM, preferably below 350 nM, more preferably below 200 nM,even more preferably below 100 nM, or most preferably below 50.0 nM.166. The derivative of any of the previous embodiments, wherein thebinding affinity to the GLP-1 receptor is measured by way ofdisplacement of ¹²⁵I-GLP-1 from the receptor, preferably using a SPAbinding assay.167. The derivative of any of the previous embodiments, wherein theGLP-1 receptor is prepared using a stable, transfected cell line,preferably a hamster cell line, more preferably a baby hamster kidneycell line, such as BHK tk-ts13.168. The derivative of any of the previous embodiments, wherein the IC₅₀value is determined as the concentration which displaces 50% of¹²⁵I-GLP-1 from the receptor.169. The derivative of any of the previous embodiments, which has anoral bioavailability, preferably an absolute oral bioavailability, whichis higher than that of semaglutide.170. The derivative of any of the previous embodiments, wherein oralbioavailability is measured in vivo in rats, as exposure in plasma afterdirect injection into the intestinal lumen.171. The derivative of any of the previous embodiments, for which theplasma concentration (pM) of the derivative, determined 30 minutes afterinjection of a solution of the derivative in the jejunum of rat, dividedby the concentration (pM) of the injected solution (dose-correctedexposure at 30 min) is at least 39, or at least 40; preferably at least60; more preferably at least 80; still more preferably at least 100;even more preferably at least 125; or most preferably at least 150.172. The derivative of any of the previous embodiments, for which theplasma concentration (pM) of the derivative, determined 30 minutes afterinjection of a solution of the derivative in the jejunum of rat, dividedby the concentration (pM) of the injected solution (dose-correctedexposure at 30 min) is at least 160, preferably at least 180, morepreferably at least 200, or most preferably at least 250.173. The derivative of any of the previous embodiments, wherein theGLP-1 derivative is tested in a concentration of 1000 uM in admixturewith 55 mg/ml sodium caprate.174. The derivative of any of the previous embodiments, wherein maleSprague Dawley rats are used, preferably with a body weight upon arrivalof approximately 240 g.175. The derivative of any of the previous embodiments, wherein the ratsare fasted for approximately 18 hours before the experiment.176. The derivative of any of the previous embodiments, wherein the ratsare taken into general anaesthesia after having fasted and before theinjection of the derivative in the jejunum.177. The derivative of any of the previous embodiments, wherein thederivative is administered in the proximal part of the jejunum (10 cmdistal for the duodenum), or in the mid-intestine (50 cm proximal forthe cecum).178. The derivative of any of the previous embodiments, wherein 100 μlof the derivative is injected into the jejunal lumen through a catheterwith a syringe, and subsequently 200 μl of air is pushed into thejejunal lumen with another syringe, which is then left connected to thecatheter to prevent flow back into the catheter.179. The derivative of any of the previous embodiments, wherein bloodsamples (200 ul) are collected into EDTA tubes from the tail vein atdesired intervals, such as at times 0, 10, 30, 60, 120 and 240 min, andcentrifuged 5 minutes, 10000 G, at 4° C. within 20 minutes.180. The derivative of any of the previous embodiments, wherein plasma(e.g. 75 ul) is separated, immediately frozen, and kept at −20° C. untilanalyzed for plasma concentration of the derivative.181. The derivative of any of the previous embodiments, wherein LOCI(Luminescent Oxygen Channeling Immunoassay) is used for analyzing theplasma concentration of the derivative.182. The derivative of any of the previous embodiments, wherein thederivative is effective at lowering blood glucose in vivo in db/db mice.183. The derivative of any of the previous embodiments, wherein thederivative is effective at lowering body weight in vivo in db/db mice.184. The derivative of any of the previous embodiments, wherein db/dbmice are treated, s.c., with a suitable range of doses of the GLP-1derivative, and blood glucose and/or bodyweight is/are determined atappropriate intervals.185. The derivative of any of the previous embodiments, wherein the doseof the GLP-1 derivative is 0.3 nmol/kg, 1.0 nmol/kg, 3.0 nmol/kg, 10nmol/kg, 30 nmol/kg, and 100 nmol/kg, wherein kg refers to the bodyweight of the mouse.186. The derivative of any of the previous embodiments, wherein acontrol group is treated with vehicle, s.c., preferably the medium inwhich the GLP-1 derivative is dissolved, e.g. with the followingcomposition: 50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween80, pH 7.4.187. The derivative of any of the previous embodiments, wherein bloodglucose is determined, and/or the mice are weighed, at time −½ h (halfan hour prior to dosing (t=0)), and at times 1, 2, 4, and 8 h.188. The derivative of any of the previous embodiments, wherein theglucose concentration is measured using the glucose oxidase method.189. The derivative of any of the previous embodiments, wherein

-   -   (i) ED₅₀ (body weight (BW)) is calculated as the dose giving        rise to half-maximum effect on delta (e.g., decrease) BW 8 hours        following the subcutaneous administration of the derivative;        and/or    -   (ii) ED₅₀ (blood glucose (BG)) is calculated as the dose giving        rise to half-maximum effect on AUC (Area Under the Curve) delta        (e.g., decrease) BG 8 hours and/or 24 hours following the        subcutaneous administration of the derivative.        190. The derivative of any of the previous embodiments, wherein        a sigmoidal dose-response relationship exists, preferably with a        clear definition of the maximum response.        191. The derivative of any of the previous embodiments, which        has a more protracted profile of action than liraglutide.        192. The derivative of any of the previous embodiments, wherein        protraction means half-life in vivo in a relevant animal        species, such as db/db mice, rat, pig, and/or, preferably,        minipig; wherein the derivative is administered i) s.c.,        and/or, ii) i.v.; preferably ii) i.v.        193. The derivative of any of the previous embodiments, wherein        the terminal half-life (T ) after i.v. administration in        minipigs is        a) at least 12 hours, preferably at least 24 hours, more        preferably at least 36 hours, even more preferably at least 48        hours, or most preferably at least 60 hours;        b) at least 7 hours, preferably at least 16 hours, more        preferably at least 24 hours, even more preferably at least 30        hours, or most preferably at least 40 hours;        c) at least 50 hours, preferably at least 60 hours, more        preferably at least 70 hours, even more preferably at least 80        hours, or most preferably at least 90 hours.        194. The derivative of any of the previous embodiments, wherein        the minipigs are male Göttingen minipigs.        195. The derivative of any of the previous embodiments, wherein        the minipigs are 7-14 months of age, and preferably weighing        from 16-35 kg.        196. The derivative of any of the previous embodiments, wherein        the minipigs are housed individually, and fed once or twice        daily, preferably with SDS minipig diet.        197. The derivative of any of the previous embodiments, wherein        the derivative is dosed, i.v., after at least 2 weeks of        acclimatisation.        198. The derivative of any of the previous embodiments, wherein        the animals are fasted for approximately 18 h before dosing and        for at least 4 h after dosing, and have ad libitum access to        water during the whole period.        199. The derivative of any of the previous embodiments, wherein        the GLP-1 derivative is dissolved in 50 mM sodium phosphate, 145        mM sodium chloride, 0.05% tween 80, pH 7.4 to a suitable        concentration, preferably from 20-60 nmol/ml.        200. The derivative of any of the previous embodiments, wherein        intravenous injections of the derivative are given in a volume        corresponding to 1-2 nmol/kg.        201. The derivative of any of the previous embodiments, which        increases the glucose stimulated insulin secretion in minipigs.        202. The derivative of any of the previous embodiments, wherein        the minipigs are male Göttingen minipigs.        203. The derivative of any of the previous embodiments, wherein        the minipigs are 7-14 months of age.        204. The derivative of any of the previous embodiments, wherein        the minipigs are housed in single pens, and fed once or twice        daily, preferably with SDS minipig fodder.        205. The derivative of any of the previous embodiments, wherein        a single dose, optionally after a period with dose escalation,        is given i.v., or s.c., in the thin skin behind the ear.        206. The derivative of any of the previous embodiments, wherein        the animals are fasted for approximately 18 h before dosing.        207. The derivative of any of the previous embodiments, wherein        a baseline group and a number of derivative dose groups        corresponding to 2-6 different plasma concentration levels are        tested, wherein the baseline group is a) vehicle treated, or b)        untreated.        208. The derivative of any of the previous embodiments, wherein        the plasma concentration level is 3000-80000 pM.        209. The derivative of any of the previous embodiments, wherein        a 1 or 2 hour intravenous glucose tolerance test (IVGTT) is        performed.        210. The derivative of any of the previous embodiments, wherein        0.3 g/kg glucose is given i.v. over a period of 30 seconds, and        blood samples taken at suitable time points, such as the        following time points (t=0 corresponds to the glucose bolus):        −10, −5, 0, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90,        100, 110, 120 minutes.        211. The derivative of any of the previous embodiments, wherein        the concentration in plasma of the derivative, glucose, and        insulin is determined.        212. The derivative of any of the previous embodiments, wherein        the derivative concentration is measured at t=0 min, and,        optionally, at the end of the test (t=60 min, or t=120 min).        213. The derivative of any of the previous embodiments, wherein        glucose is analyzed using the glucose oxidase method.        214. The derivative of any of the previous embodiments, wherein        the area under the insulin curve (AUCinsulin) is calculated and        used as a measure of insulin secretion.        215. The derivative of any of the previous embodiments, wherein        for at least one concentration thereof, the AUCinsulin is higher        than the baseline AUCinsulin, preferably at least 110% thereof,        more preferably at least 120% thereof, even more preferably at        least 130% thereof or most preferably at least 140% thereof.        216. The derivative of any of the previous embodiments, which        causes a reduced food intake in pigs relative to a control        (preferably vehicle-treated, or untreated);

optionally the food intake (0-24 h) may be 90% or lower relative to thevehicle-treated control, preferably 80% or lower, more preferably 70% orlower, even more preferably 60% or lower, or most preferably 50% orlower;

wherein food intake (0-24 h) refers to the first 24 hours afteradministration of the derivative or vehicle.

217. The derivative of any of the previous embodiments, wherein the pigsare female Landrace Yorkshire Duroc (LYD) pigs.218. The derivative of any of the previous embodiments, wherein the pigsare 3 months of age, and preferably have a weight of 30-35 kg.219. The derivative of any of the previous embodiments, wherein theanimals are housed in a group for 1-2 weeks for acclimatisation.220. The derivative of any of the previous embodiments, wherein duringthe experimental period the animals are placed in individual pens fromMonday morning to Friday afternoon for measurement of individual foodintake.221. The derivative of any of the previous embodiments, wherein theanimals are fed ad libitum with pig fodder (such as Svinefoder,Antonio).222. The derivative of any of the previous embodiments, wherein foodintake is monitored on line by logging the weight of fodder every 15minutes, preferably using the Mpigwin system.223. The derivative of any of the previous embodiments, which is dosed0.3, 1.0, 3.0, 10, or 30 nmol/kg, preferably dissolved in a phosphatebuffer (50 mM phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 8),more preferably at concentrations of 12, 40, 120, 400, or 1200 nmol/ml.224. The derivative of any of the previous embodiments, wherein thephosphate buffer serves as vehicle.225. The derivative of any of the previous embodiments, wherein theanimals are dosed with a single subcutaneous dose of the derivative, orvehicle (preferably with a dose volume of 0.025 ml/kg), on the morningof day 1, and food intake is measured for 4 days after dosing.226. The derivative of any of the previous embodiments, which has ahalf-life (T ) in vivo in rats after i.v. administration of at least 4hours, preferably at least 6 hours, even more preferably at least 8hours, or most preferably at least 10 hours.227. The derivative of any of the previous embodiments, which has ahalf-life (T ) in vivo in rats after i.v. administration of at least 12hours, preferably at least 15 hours, even more preferably at least 18hours, or most preferably at least 20 hours.228. The derivative of any of the previous embodiments, which has ahalf-life (T ) in vivo in rats after i.v. administration of at least 24hours, preferably at least 26 hours, or most preferably at least 30hours.229. The derivative of any of the previous embodiments, in which therats are male Sprague Dawley rats with a body weight of approximately400 g.230. An intermediate product in the form of a GLP-1 analogue whichcomprises the following change as compared to GLP-1(7-37) (SEQ ID NO:1): (i) 38Q; and/or (ii) 39G; or a pharmaceutically acceptable salt,amide, or ester thereof.231. The GLP-1 analogue of embodiment 230 which comprises (38E, 39G).232. An intermediate product in the form of a GLP-1 analogue whichcomprises, preferably has, the following amino acid changes, as comparedto GLP-1(7-37) (SEQ ID NO: 1): (i) 22E, 26R, 27K, 34R, 37K; (ii) 22E,26R, 27K, 30E, 34R, 36K, 38E, 39G; (iii) 22E, 26R, 27K, 34R, 36K, des37;(iv) 22E, 25V, 26R, 27K, 34R, 37K; (v) 8Aib, 20K, 22E, 26R, 27K, 30E,34G, des35-37; (vi) 26R, 27K, 30E, 34R, 36K, 38E; (vii) 8Aib, 22K, 25V,26R, 27K, 31H, 34R; (iix) 8Aib, 22K, 25V, 26R, 27K, 34R, des35-37; (ix)8Aib, 22K, 25V, 26R, 27K, 34R, des36-37; (x) 26H, 27K, 30E, 34R, 36K,38E; (xi) 22K, 25V, 26R, 27K, 30E, 34Q; (xii) 25V, 26R, 27K, 30E, 34R,36K, 38Q; (xiii) 25V, 26R, 27K, 30E, 34Q, 36K, 38E; (xiv) 22K, 26R, 27K,31H, 34G, des35-37; (xv) 8Aib, 25V, 26R, 27K, 31H, 34Q, 37K; (xvi) 25V,26R, 27K, 31H, 34Q, 37K; (xvii) 22E, 23E, 25V, 26R, 27K, 31H, 34Q, 37K;(iixx) 8Aib, 12K, 22E, 26R, 27K, 31H, 34Q; (ixx) 8Aib, 22K, 26R, 27K,31H, 34G, des35-37; (xx) 22E, 26H, 27K, 30E, 34R, 36K, 38E; (xxi) 22E,24K, 26R, 27K, 31H, 34G, des35-37; (xxii) 25V, 26R, 27K, 34Q, 36K;(xxiii) 22E, 24K, 25V, 26R, 27K, 31H, 34R; (xxiv) 22E, 24K, 25V, 26R,27K, 34G, des35-37; (xxv) 22E, 24K, 25V, 26R, 27K, 34R; or (xxvi) 8Aib,22E, 24K, 25V, 26R, 27K, 31H, 34Q; or a pharmaceutically acceptablesalt, amide, or ester thereof.233. A derivative according to any of the previous embodiments, for useas a medicament.234. A derivative according to any of the previous embodiments, for usein the treatment and/or prevention of all forms of diabetes and relateddiseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving 3-cell function, and/or for delaying or preventing diabeticdisease progression.235. A method for treating or preventing all forms of diabetes andrelated diseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving 3-cell function, and/or for delaying or preventing diabeticdisease progression—by administering a pharmaceutically active amount ofa derivative according to any of the previous embodiments.

The following are still further particular embodiments of the invention:

1. A derivative of a GLP-1 analogue,

which analogue comprises a first K residue at a position correspondingto position 27 of GLP-1(7-37) (SEQ ID NO: 1); a second K residue at aposition corresponding to position T of GLP-1(7-37), where T is aninteger in the range of 7-37 except 18 and 27; and a maximum of tenamino acid changes as compared to GLP-1(7-37); wherein the first Kresidue is designated K²⁷, and the second K residue is designated K^(T);

which derivative comprises two protracting moieties attached to K²⁷ andK^(T), respectively, via a linker, wherein

the protracting moiety is selected from Chem. 1 and Chem. 2:

HOOC—(CH₂)_(x)—CO—*  Chem. 1:

HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:

in which x is an integer in the range of 6-18, and y is an integer inthe range of 3-17; and

the linker comprises Chem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5;

or a pharmaceutically acceptable salt, amide, or ester thereof.

2. The derivative of embodiment 1, wherein the linker further comprisesa Glu di-radical selected from Chem. 6, and/or Chem. 7:

3. The derivative of any of the previous embodiments, wherein the linkeris attached to the epsilon-amino group of the first or the second Kresidue.4. The derivative of any of the previous embodiments, wherein T is 12,20, 22, 24, 36, or 37.5. The derivative of any of the previous embodiments, wherein theanalogue comprises no K residues other than the first and the second Kresidue.6. The derivative of any of the previous embodiments, wherein x is 12.7. The derivative of any of the previous embodiments, wherein y is 9.8. The derivative of any of the previous embodiments, wherein k is 1.9. The derivative of any of the previous embodiments, wherein n is 1.10. The derivative of any of the previous embodiments, wherein theanalogue comprises a GLP-1 analogue of Formula I (SEQ ID NO: 3):

Xaa₇-Xaa₈-Glu-Gly-Thr-Xaa₁₂-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Xaa₂₄-Xaa₂₅-Xaa₂₆-Lys-Phe-Ile-Xaa₃₀-Xaa₃₁-Leu-Val-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉,  FormulaI:

wherein

Xaa₇ is L-histidine, imidazopropionyl, α-hydroxy-histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, N^(α)-acetyl-histidine, N^(α)-formyl-histidine,α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine,2-pyridylalanine or 4-pyridylalanine;

Xaa₈ is Ala, Gly, Val, Leu, lie, Thr, Ser, Lys, Aib,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or(1-aminocyclooctyl) carboxylic acid;

Xaa₁₂ is Lys or Phe;

Xaa₁₆ is Val or Leu;

Xaa₁₈ is Ser, Arg, Asn, Gin, or Glu;

Xaa₁₉ is Tyr or Gin;

Xaa₂₀ is Leu, Lys, or Met;

Xaa₂₂ is Gly, Glu, Lys, or Aib;

Xaa₂₃ is Gin, Glu, or Arg;

Xaa₂₄ is Ala or Lys;

Xaa₂₅ is Ala or Val;

Xaa₂₆ is Val, His, or Arg;

Xaa₃₀ is Ala, Glu, or Arg;

Xaa₃₁ is Trp or His;

Xaa₃₄ is Glu, Asn, Gly, Gin, or Arg;

Xaa₃₅ is Gly, Aib, or absent;

Xaa₃₆ is Arg, Gly, Lys, or absent;

Xaa₃₇ is Gly, Ala, Glu, Pro, Lys, Arg, or absent;

Xaa₃₈ is Ser, Gly, Ala, Glu, Gin, Pro, Arg, or absent; and

Xaa₃₉ is Gly or absent.

11. A compound according to any of the previous embodiments, selectedfrom the following: Chem. 50, Chem. 51, Chem. 52, Chem. 53, Chem. 54,Chem. 55, Chem. 56, Chem. 57, Chem. 58, Chem. 59, Chem. 60, Chem. 61,Chem. 62, Chem. 63, Chem. 64, Chem. 65, Chem. 66, Chem. 67, Chem. 68,Chem. 69, Chem. 70, Chem. 71, Chem. 72, Chem. 73, Chem. 74, Chem. 75,Chem. 76, Chem. 77, Chem. 78, and Chem. 79; or a pharmaceuticallyacceptable salt, amide, or ester thereof.12. An intermediate product in the form of a GLP-1 analogue whichcomprises the following change as compared to GLP-1(7-37) (SEQ ID NO:1): (i) 38Q; and/or (ii) 39G; or a pharmaceutically acceptable salt,amide, or ester thereof.13. A derivative according to any of embodiments 1-11, for use as amedicament.14. A derivative according to any of embodiments 1-11, for use in thetreatment and/or prevention of all forms of diabetes and relateddiseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving 3-cell function, and/or for delaying or preventing diabeticdisease progression.15. A method for treating or preventing all forms of diabetes andrelated diseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression—by administering a pharmaceutically active amount ofa derivative according to any of embodiments 1-11.

EXAMPLES

This experimental part starts with a list of abbreviations, and isfollowed by a section including general methods for synthesising andcharacterising analogues and derivatives of the invention. Then followsa number of examples which relate to the preparation of specific GLP-1derivatives, and at the end a number of examples have been includedrelating to the activity and properties of these analogues andderivatives (section headed pharmacological methods).

The examples serve to illustrate the invention.

Abbreviations

The following abbreviations are used in the following, in alphabeticalorder:

-   Aib: aminoisobutyric acid (α-aminoisobutyric acid)-   API: Active Pharmaceutical Ingredient-   AUC: Area Under the Curve-   BG: Blood Glucose-   BHK Baby Hamster Kidney-   BW: Body Weight-   Boc: t-butyloxycarbonyl-   BSA: Bovine serum albumin-   collidine: 2,4,6-trimethylpyridine-   DCM: dichloromethane-   Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-   DIC: diisopropylcarbodiimide-   DIPEA: diisopropylethylamine-   DMAP: 4-dimethylaminopyridine-   DMEM: Dulbecco's Modified Eagle's Medium (DMEM)-   EDTA: ethylenediaminetetraacetic acid-   EGTA: ethylene glycol tetraacetic acid-   FCS: Fetal Calf Serum-   Fmoc: 9-fluorenylmethyloxycarbonyl-   HATU: (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate)-   HBTU: (2-(1H-benzotriazol-1-yl-)-1,1,3,3 tetramethyluronium    hexafluorophosphate)-   HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-   HFIP 1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol-   HOAt: 1-hydroxy-7-azabenzotriazole-   HOBt: 1-hydroxybenzotriazole-   HPLC: High Performance Liquid Chromatography-   HSA: Human Serum Albumin-   IBMX: 3-isobutyl-1-methylxanthine-   Imp: Imidazopropionic acid (also referred to as des-amino histidine,    DesH)-   i.v. intravenously-   ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl-   IVGTT: Intravenous Glucose Tolerance Test-   LCMS: Liquid Chromatography Mass Spectroscopy-   LYD: Landrace Yorkshire Duroc-   MALDI-MS: See MALDI-TOF MS-   MALDI-TOF MS: Matrix-Assisted Laser Desorption/lonisation Time of    Flight Mass Spectroscopy-   MeOH: methanol-   Mmt: 4-methoxytrityl-   Mtt: 4-methyltrityl-   NMP: N-methyl pyrrolidone-   OBz: benzoyl ester-   OEG: 8-amino-3,6-dioxaoctanic acid-   OtBu: tert-butyl ester-   PBS: Phosphate Buffered Saline-   PD: Pharmacodynamic-   Pen/Strep: Pencillin/Streptomycin-   PK: Pharmacokinetic-   RP: Reverse Phase-   RP-HPLC: Reverse Phase High Performance Liquid Chromatography-   RT: Room Temperature-   Rt: Retention time-   s.c.: Subcutaneously-   SD: Standard Deviation-   SEC-HPLC: Size Exclusion High Performance Liquic Chromatography-   SEM: Standard Error of Mean-   SPA: Scintillation Proximity Assay-   SPPS: Solid Phase Peptide Synthesis-   tBu: tert-butyl-   TFA: trifluoroacetic acid-   TIS: triisopropylsilane-   Tris: tris(hydroxymethyl)aminomethane or    2-amino-2-hydroxymethyl-propane-1,3-diol-   Trt: triphenylmethyl or trityl-   Trx: tranexamic acid-   UPLC: Ultra Performance Liquid Chromatography

Methods of Preparation A. General Methods

This section relates to methods for solid phase peptide synthesis (SPPSmethods, including methods for de-protection of amino acids, methods forcleaving the peptide from the resin, and for its purification), as wellas methods for detecting and characterising the resulting peptide (LCMS,MALDI, and UPLC methods). The solid phase synthesis of peptides may insome cases be improved by the use of di-peptides protected on thedi-peptide amide bond with a group that can be cleaved under acidicconditions such as, but not limited to, 2-Fmoc-oxy-4-methoxybenzyl, or2,4,6-trimethoxybenzyl. In cases where a serine or a threonine ispresent in the peptide, pseudoproline di-peptides may be used (availablefrom, e.g., Novabiochem, see also W. R. Sampson (1999), J. Pep. Sci., 5,403). The protected amino acid derivatives used were standard Fmoc-aminoacids (supplied from e.g. Anaspec, IRIS, or Novabiochem). The N-terminalamino acid was Boc protected at the alpha amino group (e.g.Boc-His(Boc)-OH, or Boc-His(Trt)-OH for peptides with His at theN-terminus). The epsilon amino group of lysines in the sequence wereeither protected with Mtt, Mmt, Dde, ivDde, or Boc, depending on theroute for attachment of the albumin binding moiety and spacer. Thealbumin binding moiety and/or linker can be attached to the peptideeither by acylation of the resin bound peptide or by acylation insolution of the unprotected peptide. In case of attachment of thealbumin binding moiety and/or linker to the protected peptidyl resin,the attachment can be modular using SPPS and suitably protected buildingblocks such as but not limited to Fmoc-Oeg-OH(Fmoc-8-amino-3,6-dioxaoctanoic acid), Fmoc-Trx-OH (Fmoc-tranexamicacid), Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester,nonadecanedioic acid mono-tert-butyl ester, or 4-(9-carboxynonyloxy)benzoic acid tert-butyl ester.

1. Synthesis of Resin Bound Peptide SPPS Method B

SPPS method B refers to the synthesis of a protected peptidyl resinusing Fmoc chemistry on a microwave-based Liberty peptide synthesiser(CEM Corp., North Carolina). A suitable resin is a pre-loaded, low-loadWang resin available from Novabiochem (e.g. low load Fmoc-Lys(Mtt)-Wangresin, 0.35 mmol/g). Fmoc-deprotection was with 5% piperidine in NMP atup to 70 or 75° C. The coupling chemistry was DIC/HOAt in NMP. Aminoacid/HOAt solutions (0.3 M in NMP at a molar excess of 3-10 fold) wereadded to the resin followed by the same molar equivalent of DIC (0.75Min NMP). For example, the following amounts of 0.3 M amino acid/HOAtsolution were used per coupling for the following scale reactions:Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml, 1 mmol/15 ml. Coupling timesand temperatures were generally 5 minutes at up to 70 or 75° C. Longercoupling times were used for larger scale reactions, for example 10 min.Histidine amino acids were double coupled at 50° C., or quadruplecoupled if the previous amino acid was sterically hindered (e.g. Aib).Arginine amino acids were coupled at RT for 25 min then heated to 70 or75° C. for 5 min. Some amino acids such as but not limited to Aib, were“double coupled”, meaning that after the first coupling (e.g. 5 min at75° C.), the resin is drained and more reagents are added (amino acid,HOAt and DIC), and the mixture is heated again (e.g. 5 min at 75° C.).When a chemical modification of a lysine side chain was desired, thelysine was incorporated as Lys(Mtt). The Mtt group was removed bywashing the resin with DCM and suspending the resin in neat (undiluted)hexafluoroisopropanol for 20 minutes followed by washing with DCM andNMP. The chemical modification of the lysine was performed either bymanual synthesis (see SPPS method D) or by one or more automated stepson the Liberty peptide synthesiser as described above, using suitablyprotected building blocks (see General methods), optionally including amanual coupling.

SPPS Method D

SPPS method D refers to synthesis of the protected peptidyl resin usingmanual Fmoc chemistry. This was typically used for the attachment of thelinkers and side chains to the peptide backbone. The followingconditions were employed at 0.25 mmol synthesis scale. The couplingchemistry was DIC/HOAt/collidine in NMP at a 4-10 fold molar excess.Coupling conditions were 1-6 h at room temperature. Fmoc-deprotectionwas performed with 20-25% piperidine in NMP (3×20 ml, each 10 min)followed by NMP washings (4×20 ml). Dde- or ivDde-deprotection wasperformed with 2% hydrazine in NMP (2×20 ml, each 10 min) followed byNMP washings (4×20 ml). Mtt- or Mmt-deprotection was performed with 2%TFA and 2-3% TIS in DCM (5×20 ml, each 10 min) followed by DCM (2×20ml), 10% MeOH and 5% DIPEA in DCM (2×20 ml) and NMP (4×20 ml) washings,or by treatment with neat hexafluroisopropanol (5×20 ml, each 10 min)followed by washings as above. The albumin binding moiety and/or linkercan be attached to the peptide either by acylation of the resin boundpeptide or acylation in solution of the unprotected peptide (see theroutes described below). In case of attachment of the albumin bindingmoiety and/or linker to the protected peptidyl resin the attachment canbe modular using SPPS and suitably protected building blocks (seeGeneral methods).

Attachment to Resin Bound Peptide—Route I:

Activated (active ester or symmetric anhydride) albumin binding moietyor linker such as octadecanedioic acid mono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi et al. EP511600, 4 molar equivalents relative to resinbound peptide) was dissolved in NMP (25 ml), added to the resin andshaken overnight at room temperature. The reaction mixture was filteredand the resin was washed extensively with NMP, DCM, 2-propanol, methanoland diethyl ether.

Attachment to Resin Bound Peptide—Route II:

The albumin binding moiety was dissolved in NMP/DCM (1:1, 10 ml). Theactivating reagent such as HOBt (4 molar equivalents relative to resin)and DIC (4 molar equivalents relative to resin) was added and thesolution was stirred for 15 min. The solution was added to the resin andDIPEA (4 molar equivalents relative to resin) was added. The resin wasshaken 2 to 24 hours at room temperature. The resin was washed with NMP(2×20 ml), NMP/DCM (1:1, 2×20 ml) and DCM (2×20 ml).

Attachment to Peptide in Solution—Route III:

Activated (active ester or symmetric anhydride) albumin binding moietyor linker such as octadecanedioic acid mono-(2,5-dioxo-pyrrolidin-1-yl)ester (Ebashi et al. EP511600) 1-1.5 molar equivalents relative to thepeptide was dissolved in an organic solvent such as acetonitrile, THF,DMF, DMSO or in a mixture of water/organic solvent (1-2 ml) and added toa solution of the peptide in water (10-20 ml) together with 10 molarequivalents of DIPEA. In case of protecting groups on the albuminbinding residue such as tert-butyl, the reaction mixture was lyophilisedovernight and the isolated crude peptide deprotected afterwards. In caseof tert-butyl protection groups the deprotection was performed bydissolving the peptide in a mixture of trifluoroacetic acid, water andtriisopropylsilane (90:5:5). After 30 min the mixture was evaporated invacuo and the crude peptide purified by preparative HPLC as describedlater.

SPPS Method E

SPPS method E refers to peptide synthesis by Fmoc chemistry on a PreludeSolid Phase Peptide Synthesiser from Protein Technologies (Tucson, Ariz.85714 U.S.A.). A suitable resin is a pre-loaded, low-load Wang resinavailable from Novabiochem (e.g. low load fmoc-Lys(Mtt)-Wang resin, 0.35mmol/g). Fmoc-deprotection was with 25% piperidine in NMP for 2×10 min.The coupling chemistry was DIC/HOAt/collidine in NMP. Amino acid/HOAtsolutions (0.3 M in NMP at a molar excess of 3-10 fold) were added tothe resin followed by the same molar equivalent of DIC (3 M in NMP) andcollidine (3 M in NMP). For example, the following amounts of 0.3 Mamino acid/HOAt solution were used per coupling for the following scalereactions: Scale/ml, 0.10 mmol/2.5 ml, 0.25 mmol/5 ml. Coupling timeswere generally 60 minutes. Some amino acids including, but not limitedto arginine, Aib or histidine were “double coupled”, meaning that afterthe first coupling (e.g. 60 min), the resin is drained and more reagentsare added (amino acid, HOAt, DIC, and collidine), and the mixtureallowed to react gain (e.g. 60 min). Some amino acids and fatty acidderivatives including but not limited to Fmoc-OEG-OH, Fmoc-Trx-OH,Fmoc-Glu-OtBu, octadecanedioic acid mono-tert-butyl ester,nonadecanedioic acid mono-tert-butyl ester, or 4-(9-carboxynonyloxy)benzoic acid tert-butyl ester were coupled for prolonged time, forexample 6 hours. When a chemical modification of a lysine side chain wasdesired, the lysine was incorporated as Lys(Mtt). The Mtt group wasremoved by washing the resin with DCM and suspending the resin inhexafluoroisopropanol/DCM (75:25) for 3×10 minutes followed by washingswith DCM, 20% piperidine and NMP. The chemical modification of thelysine was performed either by manual synthesis (see SPPS method D) orby one or more automated steps on the Prelude peptide synthesiser asdescribed above using suitably protected building blocks (see Generalmethods).

2. Cleavage of Peptide from the Resin and Purification

After synthesis the resin was washed with DCM, and the peptide wascleaved from the resin by a 2-3 hour treatment with TFA/TIS/water(95/2.5/2.5 or 92.5/5/2.5) followed by precipitation with diethylether.The peptide was dissolved in a suitable solvent (such as, e.g., 30%acetic acid) and purified by standard RP-HPLC on a C18, 5 μM column,using acetonitrile/water/TFA. The fractions were analysed by acombination of UPLC, MALDI and LCMS methods, and the appropriatefractions were pooled and lyophilised.

3. Methods for Detection and Characterisation LCMS Methods LCMS Method 1(LCMS1)

An Agilent Technologies LC/MSD TOF (G1969A) mass spectrometer was usedto identify the mass of the sample after elution from an Agilent 1200series HPLC system. The de-convolution of the protein spectra wascalculated with Agilent's protein confirmation software.

Eluents:

A: 0.1% Trifluoro acetic acid in waterB: 0.1% Trifluoro acetic acid in acetonitrile

Column: Zorbax 5u, 300SB-C3, 4.8×50 mm

Gradient: 25%-95% acetonitrile over 15 min

LCMS Method 2 (LCMS2)

A Perkin Elmer Sciex API 3000 mass spectrometer was used to identify themass of the sample after elution from a Perkin Elmer Series 200 HPLCsystem.

Eluents:

A: 0.05% Trifluoro acetic acid in waterB: 0.05% Trifluoro acetic acid in acetonitrile

Column: Waters Xterra MS C-18×3 mm id 5 μm

Gradient: 5%-90% acetonitrile over 7.5 min at 1.5 ml/min

LCMS Method 3 (LCMS3)

A Waters Micromass ZQ mass spectrometer was used to identify the mass ofthe sample after elution from a Waters Alliance HT HPLC system.

Eluents:

A: 0.1% Trifluoro acetic acid in waterB: 0.1% Trifluoro acetic acid in acetonitrile

Column: Phenomenex, Jupiter C4 50×4.60 mm id 5 μm

Gradient: 10%-90% B over 7.5 min at 1.0 ml/min

LCMS Method 4 (LCMS4)

LCMS4 was performed on a setup consisting of Waters Acquity UPLC systemand LCT Premier XE mass spectrometer from Micromass. The UPLC pump wasconnected to two eluent reservoirs containing:

A: 0.1% Formic acid in waterB: 0.1% Formic acid in acetonitrileThe analysis was performed at RT by injecting an appropriate volume ofthe sample (preferably 2-10 μl) onto the column which was eluted with agradient of A and B.The UPLC conditions, detector settings and mass spectrometer settingswere:Column: Waters Acquity UPLC BEH, C-18, 1.7 μm, 2.1 mm×50 mmGradient: Linear 5%-95% acetonitrile during 4.0 min (alternatively 8.0min) at 0.4 ml/minDetection: 214 nm (analogue output from TUV (Tunable UV detector))MS ionisation mode: API-ESScan: 100-2000 amu (alternatively 500-2000 amu), step 0.1 amu

UPLC and HPLC Methods Method 05_B5_1

UPLC (method 05_B5_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:A: 0.2 M Na₂SO₄, 0.04 M H₃PO₄, 10% CH₃CN (pH 3.5)B: 70% CH₃CN, 30% H₂OThe following linear gradient was used: 60% A, 40% B to 30% A, 70% Bover 8 minutes at a flow-rate of 0.40 ml/min.

Method 05_B7_1

UPLC (method 05_B7_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:A: 0.2 M Na₂SO₄, 0.04 M H₃PO₄, 10% CH₃CN (pH 3.5)B: 70% CH₃CN, 30% H₂OThe following linear gradient was used: 80% A, 20% B to 40% A, 60% Bover 8 minutes at a flow-rate of 0.40 ml/min.

Method 04_A2_1

UPLC (method 04_A2_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130A, 1.7 um, 2.1mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:A: 90% H₂O, 10% CH₃CN, 0.25 M ammonium bicarbonateB: 70% CH₃CN, 30% H₂OThe following linear gradient was used: 90% A, 10% B to 60% A, 40% Bover 16 minutes at a flow-rate of 0.40 ml/min.

Method 04_A3_1

UPLC (method 04_A3_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:A: 90% H₂O, 10% CH₃CN, 0.25 M ammonium bicarbonateB: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 75% A, 25% B to 45% A, 55% Bover 16 minutes at a flow-rate of 0.40 ml/min.

Method 04_A4_1

UPLC (method 04_A4_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:A: 90% H₂O, 10% CH₃CN, 0.25 M ammonium bicarbonateB: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 65% A, 35% B to 25% A, 65% Bover 16 minutes at a flow-rate of 0.40 ml/min.

Method 08_B2_1

UPLC (method 08_B2_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 99.95% H₂O, 0.05% TFA B: 99.95% CH₃CN, 0.05% TFA

The following linear gradient was used: 95% A, 5% B to 40% A, 60% B over16 minutes at a flow-rate of 0.40 ml/min.

Method 08_B4_1

UPLC (method 08_B4_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:

A: 99.95% H₂O, 0.05% TFA B: 99.95% CH₃CN, 0.05% TFA

The following linear gradient was used: 95% A, 5% B to 95% A, 5% B over16 minutes at a flow-rate of 0.40 ml/min.

Method 05_B10_1

UPLC (Method 05_B10_1): The RP-analyses was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7um, 2.1 mm×150 mm column, 40° C.

The UPLC system was connected to two eluent reservoirs containing:A: 0.2 M Na₂SO₄, 0.04 M H₃PO₄, 10% CH₃CN (pH 3.5)B: 70% CH₃CN, 30% H₂O

The following linear gradient was used: 40% A, 60% B to 20% A, 80% Bover 8 minutes at a flow-rate of 0.40 ml/min.

Method 01_A4_2

UPLC (Method 01_A4_2): The RP-analysis was performed using a Waters 600Ssystem fitted with a waters 996 diode array detector. UV detections at214 nm and 254 nm were collected using a Symmetry300 C18, 5 um, 3.9mm×150 mm column, 42° C. The HPLC system was connected to three eluentreservoirs containing: A: 100% H₂O, B: 100% CH₃CN, C: 1% trifluoroaceticacid in H₂O. The following linear gradient was used: 90% A, 5% B, 5% Cto 0% A, 95% B, 5% C over 15 minutes at a flow-rate of 1.0 ml/min.

Method 09_B2_1

UPLC (Method 09_B2_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1mm×150 mm column, 40° C. The UPLC system was connected to two eluentreservoirs containing: A: 99.95% H₂O, 0.05% TFA; B: 99.95% CH₃CN, 0.05%TFA. The following linear gradient was used: 95% A, 5% B to 40% A, 60% Bover 16 minutes at a flow-rate of 0.40 ml/min.

Method 09_B4_1

UPLC (Method 09_B4_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1mm×150 mm column, 40° C. The UPLC system was connected to two eluentreservoirs containing: A: 99.95% H₂O, 0.05% TFA; B: 99.95% CH₃CN, 0.05%TFA. The following linear gradient was used: 95% A, 5% B to 5% A, 95% Bover 16 minutes at a flow-rate of 0.40 ml/min.

Method 05_B8_1

UPLC (Method 05_B8_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1mm×150 mm column, 40° C. The UPLC system was connected to two eluentreservoirs containing: A: 0.2 M Na₂SO₄, 0.04 M H₃PO₄, 10% CH₃CN (pH3.5); B: 70% CH₃CN, 30% H₂O. The following linear gradient was used: 50%A, 50% B to 20% A, 80% B over 8 minutes at a flow-rate of 0.40 ml/min.

Method 10_B14_1

UPLC (Method 10_B14_1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH ShieldRP18, 1.7 um,2.1 mm×150 mm column, 50° C. The UPLC system was connected to two eluentreservoirs containing: A: 99.95% H₂O, 0.05% TFA; B: 99.95% CH₃CN, 0.05%TFA. The following linear gradient was used: 70% A, 30% B to 40% A, 60%B over 12 minutes at a flow-rate of 0.40 ml/min.

Method 04_A6_1

UPLC (Method 04_A6_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1mm×150 mm column, 40° C. The UPLC system was connected to two eluentreservoirs containing: A: 10 mM TRIS, 15 mM ammonium sulphate, 80% H₂O,20%, pH 7.3; B: 80% CH₃CN, 20% H₂O. The following linear gradient wasused: 95% A, 5% B to 10% A, 90% B over 16 minutes at a flow-rate of 0.35ml/min.

Method 01_B4_1

HPLC (Method 01_B4_1): The RP-analysis was performed using a Waters 600Ssystem fitted with a Waters 996 diode array detector. UV detections werecollected using a Waters 3 mm×150 mm 3.5 um C-18 Symmetry column. Thecolumn was heated to 42° C. and eluted with a linear gradient of 5-95%acetonitrile, 90-0% water, and 5% trifluoroacetic acid (1.0%) in waterover 15 minutes at a flow-rate of 1 ml/min.

Method 04_A7_1

UPLC (Method 04_A7_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1mm×150 mm column, 40° C. The UPLC system was connected to two eluentreservoirs containing: A: 10 mM TRIS, 15 mM ammonium sulphate, 80% H₂O,20%, pH 7.3; B: 80% CH₃CN, 20% H₂O. The following linear gradient wasused: 95% A, 5% B to 40% A, 60% B over 16 minutes at a flow-rate of 0.40ml/min.

Method 05_B9_1

UPLC (Method 05_B9_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH130, C18, 130 Å, 1.7 um, 2.1mm×150 mm column, 40° C. The UPLC system was connected to two eluentreservoirs containing: A: 0.2 M Na₂SO₄, 0.04 M H₃PO₄, 10% CH₃CN (pH3.5); B: 70% CH₃CN, 30% H₂O. The following linear gradient was used: 70%A, 30% B to 20% A, 80% B over 8 minutes at a flow-rate of 0.40 ml/min.

Method 10_B12_1

UPLC (Method 10_B12_1): The RP-analysis was performed using a WatersUPLC system fitted with a dual band detector. UV detections at 214 nmand 254 nm were collected using an ACQUITY UPLC BEH ShieldRP18, 1.7 um,2.1 mm×150 mm column, 50° C. The UPLC system was connected to two eluentreservoirs containing: A: 99.95% H₂O, 0.05% TFA; B: 99.95% CH₃CN, 0.05%TFA. The following linear gradient was used: 50% A, 50% B to 0% A, 100%B over 16 minutes at a flow-rate of 0.40 ml/min.

Method 04_A9_1

UPLC (Method 04_A9_1): The RP-analysis was performed using a Waters UPLCsystem fitted with a dual band detector. UV detections at 214 nm and 254nm were collected using an ACQUITY UPLC BEH Shield RP18, C18, 1.7 um,2.1 mm×150 mm column, 60° C. The UPLC system was connected to two eluentreservoirs 25 containing: A: 200 mM Na₂SO₄+20 mM Na₂HPO₄+20 mM NaH₂PO₄in 90% H₂O /10% CH₃CN, pH 7.2; B: 70% CH₃CN, 30% H₂O. The following stepgradient was used: 90% A, 10% B to 80% A, 20% B over 3 minutes, 80% A,20% B to 50% A, 50% B over 17 minutes at a flow-rate of 0.40 ml/min.

MALDI-MS Method

Molecular weights were determined using matrix-assisted laser desorptionand ionisation time-of-flight mass spectroscopy, recorded on a Microflexor Autoflex (Bruker). A matrix of alpha-cyano-4-hydroxy cinnamic acidwas used.

NMR Method

Proton NMR spectra were recorded using a Brucker Avance DPX 300 (300MHz) with tetramethylsilane as an internal standard. Chemical shifts (6)are given in ppm and splitting patterns are designated as follows: s,singlet; d, doublet; dd, double doublet; dt, double triplet t, triplet,tt, triplet of triplets; q, quartet; quint, quintet; sext, sextet; m,multiplet, and br=broad.

B. Synthesis of Intermediates 1. Synthesis of Mono Esters of FattyDiacids

Overnight reflux of the C12, C14, C16 and C18 diacids withBoc-anhydride, DMAP, and t-butanol in toluene gives predominately thet-butyl mono ester. Obtained is after work-up a mixture of mono acid,diacid and diester. Purification is carried out by washing, short plugsilica filtration and crystallisation.

B. Synthesis of Compounds of the Invention Example 1N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu²²,Arg²⁶,Lys²⁷,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 4)

Preparation method: SPPS method B

UPLC (Method 09_B2_1): Rt=12.4 min UPLC (Method: 04_A3_1): Rt=8.3 min

LCMS4: Rt=2.0 min, m/z=1659 (m/3), 1244 (m/4), 996 (m/5)

Example 2N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε36)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu²²,Arg²⁶,Lys²⁷,Glu³⁰,Arg³⁴,Lys³⁶]-GLP-1-(7-37)-peptidyl-Glu-Gly(SEQ ID NO: 5)

Preparation method: SPPS method B

UPLC (Method 09_B2_1): Rt=13.1 min

UPLC (Method 04_A7_1): Rt=6.3 min LCMS4: Rt=2.1 min, m/z=1707 (m/3),1280 (m/4), 1025 (m/5)

Example 3N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε36)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu²²,Arg²⁶,Lys²⁷,Arg³⁴,Lys³⁶],des-Gly37-GLP-1-(7-36)-peptide(SEQ ID NO: 6)

Preparation method: SPPS method B

UPLC (Method 09_B2_1): Rt=13.3 min UPLC (Method 05_B5_1): Rt=6.5 min

LCMS4: Rt=2.3 min, m/z=1607 (m/3), 1205 (m/4), 964 (m/5)

Example 4N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu²²,Val²⁵,Arg²⁶,Lys²⁷,Arg³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 7)

Preparation method: SPPS method B

UPLC (Method 09_B2_1): Rt=13.0 min UPLC (Method 04_A7_1): Rt=6.9 min

LCMS4: Rt=2.0 min, m/z=1668 (m/3), 1251 (m/4), 1001 (m/5)

Example 5N^(ε20)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Lys²⁰,Glu²²,Arg²⁶,Lys²⁷,Glu³⁰,Gly³⁴]-GLP-1-(7-34)-peptide(SEQ ID NO: 8)

Preparation method: SPPS method B

UPLC (Method 08_B4_1): Rt=9.02 min UPLC (Method 04_A6_1): Rt=4.61 min

LCMS4: Rt=2.17 min, m/z=1540 (m/3), 1155 (m/4)

Example 6N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε36)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Arg²⁶,Lys²⁷,Glu³⁰,Arg³⁴,Lys³⁶]-GLP-1-(7-37)-peptidyl-Glu(SEQ ID NO: 9)

Preparation method: SPPS method B

UPLC (Method 09_B2_1): Rt=13.0 min UPLC (Method 05_B5_1): Rt=5.6 min

LCMS4: Rt=2.2 min, m/z=1664 (m/3), 1248 (m/4), 999 (m/5)

Example 7N^(ε22)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Lys²²,Val²⁵,Arg²⁶,Lys²⁷,His³¹,Arg³⁴]-GLP-1-(7-37)-peptide(SEQ ID NO: 10)

Preparation method: SPPS method BLCMS2: Rt=4.00 min, m/z=1599 (m/3), 1199 (m/4)

UPLC (Method 08_B4_1): Rt=7.83 min UPLC (Method 05_B9_1): Rt=7.45 minExample 8N^(ε22)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Lys²²,Val²⁵,Arg²⁶,Lys²⁷,Arg³⁴]-GLP-1-(7-34)-peptide(SEQ ID NO: 11)

Preparation method: SPPS method B

UPLC (Method 10_B12_1): Rt=8.92 min

LCMS4: Rt=2.58 min, m/z=1525 (m/3), 1144 (m/4), 915 (m/5)

Example 9N^(ε22)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Lys22,Val25,Arg26,Lys27,Arg34]-GLP-1-(7-35)-peptide(SEQ ID NO: 12)

Preparation method: SPPS method EThe theoretical molecular mass of 4628 Da was confirmed by MALDI-MSUPLC (method 09_B4_1): Rt=9.29 minUPLC (method 04_A6_1): Rt=6.49 min

Example 10N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε36)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[His²⁶,Lys²⁷,Glu³⁰,Arg³⁴,Lys³⁶]-GLP-1-(7-37)-peptidyl-Glu(SEQ ID NO: 13)

Preparation method: SPPS method B

UPLC (Method 08_B2_1): Rt=12.9 min UPLC (Method 05_B5_1): Rt=5.5 min

LCMS4: Rt=2.2 min, m/z=1657 (m/3), 1243 (m/4), 995 (m/5)

Example 11N^(ε22)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Lys²²,Val²⁵,Arg²⁶,Lys²⁷,Glu³⁰,Gln³⁴]-GLP-1-(7-37)-peptide(SEQ ID NO: 14)

Preparation method: SPPS method B

UPLC (Method 08_B2_1): Rt=13.5 min

LCMS4: Rt=2.2 min, m/z=1621 (m/3), 1216 (m/4), 973 (m/5)

Example 12N^(α)(N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε36)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Val²⁵,Arg²⁶,Lys²⁷,Glu³⁰,Arg³⁴,Lys³⁶]-GLP-1-(7-37)-peptidyl)-Gln(SEQ ID NO: 15)

Preparation method: SPPS method B

UPLC (Method 09_B4_1): Rt=8.9 min UPLC (Method 05_B7_1): Rt=8.8 min

LCMS4: Rt=2.2 min, m/z=1673 (m/3), 1255 (m/4), 1004 (m/5)

Example 13N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε36)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Val²⁵,Arg²⁶,Lys²⁷,Glu³⁰,Gln³⁴,Lys³⁶]-GLP-1-(7-37)-peptidyl-Glu (SEQ ID NO: 16)

Preparation method: SPPS method B

UPLC (Method 05_B9_1): Rt=7.9 min UPLC (Method 05_B7_1): Rt=8.8 min

LCMS4: Rt=2.3 min, m/z=1663 (m/3), 1248 (m/4), 999 (m/5)

Example 14N^(ε22)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl],N^(ε27)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Lys²²,Arg²⁶,Lys²⁷,His³¹,Gly³⁴]-GLP-1-(7-34)-peptide(SEQ ID NO: 17)

Preparation method: SPPS method B

UPLC (Method 08_B4_1): Rt=8.5 min UPLC (Method 05_B7_1): Rt=8.8 min

LCMS4: Rt=2.1 min, m/z=1462 (m/3), 1097 (m/4), 878 (m/5)

Example 15N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Val²⁵,Arg²⁶,Lys²⁷,His³¹,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 18)

Preparation method: SPPS method B

UPLC (Method 08_B4_1): Rt=8.3 min UPLC (Method 05_B9_1): Rt=7.1 min

LCMS4: Rt=2.1 min, m/z=1589 (m/3), 1192 (m/4), 954 (m/5)

Example 16N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Val²⁵,Arg²⁶,Lys²⁷,His³¹,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide (SEQ ID NO: 19)

Preparation method: SPPS method B

UPLC (Method 08_B4_1): Rt=8.1 min

LCMS4: Rt=2.1 min, m/z=1585 (m/3), 1189 (m/4), 951 (m/5)

Example 17N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε37)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu²²,Glu²³,Val²⁵,Arg²⁶,Lys²⁷,His³¹,Gln³⁴,Lys³⁷]-GLP-1-(7-37)-peptide(SEQ ID NO: 20)

Preparation method: SPPS method B

UPLC (Method 08_B4_1): Rt=8.0 min UPLC (Method 05_B9_1): Rt=6.6 min

LCMS4: Rt=2.1 min, m/z=1609 (m/3), 1206 (m/4), 966 (m/5)

Example 18N^(ε12)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Lys¹²,Glu²²,Arg²⁶,Lys²⁷,His³¹,Gln³⁴]-GLP-1-(7-37)-peptide (SEQ ID NO:21)

Preparation method: SPPS method B

UPLC (Method 09_B4_1): Rt=7.66 min UPLC (Method 04_A6_1): Rt=4.09 min

LCMS4: Rt=1.83 min, m/z=1181(m/4), 945 (m/5)

Example 19N^(ε22)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Lys²²,Arg²⁶,Lys²⁷,His³¹,Gly³⁴]-GLP-1-(7-34)-peptide(SEQ ID NO: 22)

Preparation method: SPPS method B

UPLC (Method 09_B4_1): Rt=8.37 min UPLC (Method 04_A6_1): Rt=4.41 min

LCMS4: Rt=2.00 min, m/z=1466 (m/3), 1100 (m/4)

Example 20N^(ε22)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl],N^(ε27)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Lys²²,Val²⁵,Arg²⁶,Lys²⁷,Glu³⁰,Gln³⁴]-GLP-1-(7-37)-peptide(SEQ ID NO: 23)

Preparation method: SPPS Method E

UPLC (Method 09_B4_1): Rt=9.00 min UPLC (Method 04_A6_1): Rt=6.50 min

LCMS4: Rt=2.23 min, m/z=1215(m/4), 972(m/5)

Example 21N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε36)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu²²,His²⁶,Lys²⁷,Glu³⁰,Arg³⁴,Lys³⁶]-GLP-1-(7-37)-peptidyl-Glu(SEQ ID NO: 24)

Preparation method: SPPS method B

UPLC (Method 09_B4_1): Rt=8.4 min UPLC (Method 04_A6_1): Rt=9.3 min

LCMS4: Rt=2.2 min, m/z=1681 (m/3), 1261 (m/4), 1009 (m/5)

Example 22N^(ε24)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl],N^(ε27)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Glu²²,Lys²⁴,Arg²⁶, Lys²⁷, His³¹,Gly³⁴]-GLP-1-(7-34)-peptide (SEQ ID NO: 25)

Preparation method: SPPS method B

UPLC (Method 09_B4_1): Rt=8.17 min UPLC (Method 04_A6_1): Rt=4.65 min

LCMS4: Rt=1.98 min, m/z=1481 (m/3), 1111 (m/4)

Example 23N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε36)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Val²⁵,Arg²⁶,Lys²⁷,Gln³⁴,Lys³⁶]-GLP-1-(7-37)-peptide(SEQ ID NO: 26)

Preparation method: SPPS method B

UPLC (Method 08_B4_1): Rt=8.82 min UPLC (Method 05_B5_1): Rt=6.10 min

LCMS4: Rt=2.37 min, m/z=1687 (m/3), 1266 (m/4), 1013 (m/5)

Example 24N^(ε24)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu²²,Lys²⁴,Val²⁵,Arg²⁶,Lys²⁷,His³¹,Arg³⁴]-GLP-1-(7-37)-peptide(SEQ ID NO: 27)

Preparation method: SPPS method B

UPLC (Method 08_B4_1): Rt=7.52 min UPLC (Method 04_A9_1): Rt=10.35 min

LCMS4: Rt=1.92 min, m/z=1613 (m/3), 1210 (m/4), 968 (m/5), 807 (m/6)

Example 25N^(ε24)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu²²,Lys²⁴,Arg²⁶,Lys²⁷,His³¹,Gly³⁴]-GLP-1-(7-34)-peptide(SEQ ID NO: 28)

Preparation method: SPPS method B

UPLC (Method 08_B4_1): Rt=8.01 min UPLC (Method 04_A9_1): Rt=8.00 min

LCMS4: Rt=2.08 min, m/z=1513 (m/3), 1135 (m/4), 908 (m/5)

Example 26N^(ε27)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4R)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl],N^(ε36)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-[[(4R)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Val²⁵,Arg²⁶,Lys²⁷,Gln³⁴,Lys³⁶]-GLP-1-(7-37)-peptide(SEQ ID NO: 29)

Preparation method: SPPS method B

UPLC (Method 09_B4_1): Rt=8.96 min UPLC (Method 05_B5_1): Rt=6.54 minUPLC (Method 04_A6_1): Rt=5.69 min

LCMS4: m/z: Rt=3.07 min, m/z=1687 (m/3), 1266 (m/4), 1013 (m/5)

Example 27N^(ε24)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu²²,Lys²⁴,Val²⁵,Arg²⁶,Lys²⁷,Gly³⁴]-GLP-1-(7-34)-peptide(SEQ ID NO: 30)

Preparation method: SPPS method B

UPLC (Method 09_B4_1): Rt=9.25 min UPLC (Method 04_A6_1): Rt=6.01 min

LCMS4: Rt=3.31 min, m/z=1506 (m/3), 1130 (m/4), 4520 (m/5)

Example 28N^(ε24)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu²²,Lys²⁴,Val²⁵,Arg²⁶,Lys²⁷,Arg³⁴]-GLP-1-(7-37)-peptide(SEQ ID NO: 31)

Preparation method: SPPS method B

UPLC (Method 08_B4_1): Rt=8.19 min UPLC (Method 04_A6_1): Rt=5.24 min

LCMS4: Rt=3.22 min, m/z=1663 (m/3), 1247 (m/4), 998 (m/5), 832 (m/6)

Example 29N^(ε24)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(13-carboxytridecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Glu²²,Lys²⁴,Val²⁵,Arg²⁶,Lys²⁷,His³¹,Gln³⁴]-GLP-1-(7-37)-peptide(SEQ ID NO: 32)

Preparation method: SPPS method B

UPLC (Method 08_B4_1): Rt=7.79 min UPLC (Method 04_A6_1): Rt=4.87 minExample 30N^(ε24)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl],N^(ε27)-[(4S)-4-carboxy-4-[[2-[2-[2-[[2-[2-[2-(13-carboxytridecanoylamino)ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]butanoyl]-[Glu²²,Lys²⁴,Val²⁵,Arg²⁶,Lys²⁷,Gly³⁴]-GLP-1-(7-34)-peptide(SEQ ID NO: 33)

Preparation method: SPPS method B

UPLC (Method 08_B4_1): Rt=9.33 min UPLC (Method 04_A6_1): Rt=6.13 min

LCMS4: Rt=2.98 min, m/z=1506 (m/3), 1130 (m/4), 904 (m/5)

Example 31N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε36)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[10-(4-carboxyphenoxy)decanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Aib⁸,Glu²²,Arg²⁶,Lys²⁷,Glu30,Arg³⁴,Lys³⁶]-GLP-1-(7-37)-peptidyl-Glu-Gly(SEQ ID NO: 34)

Preparation method: SPPS ,method B

UPLC (Method 09_B4_1):_Rt=8.58 min UPLC (Method 10_B29_1): Rt=10.6 minUPLC (Method 04_A6_1): Rt=4.43 min

LCMS4: Rt=3.72 min; m/3: 1712; m/4: 1284; m/5: 1028

Example 32N^(ε27)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl],N^(ε36)-[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-[12-(4-carboxyphenoxy)dodecanoylamino]butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[Glu²²,Arg²⁶,Lys²⁷,Glu³⁰,Arg³⁴,Lys³⁶]-GLP-1-(7-37)-peptidyl-Glu-Gly(SEQ ID NO: 35)

Preparation method: SPPS method B

UPLC (Method 09_B4_1): Rt=9.19 min UPLC (Method 10_B29_1): Rt=13.73 minUPLC (Method 04_A6_1): Rt=5.40 min

LCMS4: Rt=2.44 min; m/3: 1726; m/4: 1294; m/5:1036

Pharmacological Methods Example 33: In Vitro Potency

The purpose of this example is to test the activity, or potency, of theGLP-1 derivatives in vitro.

The potencies of the GLP-1 derivatives of Examples 1-32 were determinedas described below, i.e. as the stimulation of the formation of cyclicAMP (cAMP) in a medium containing membranes expressing the human GLP-1receptor.

Principle

Purified plasma membranes from a stable transfected cell line,BHK467-12A (tk-ts13), expressing the human GLP-1 receptor werestimulated with the GLP-1 analogue or derivative in question, and thepotency of cAMP production was measured using the AlphaScreen™ cAMPAssay Kit from Perkin Elmer Life Sciences. The basic principle of theAlphaScreen Assay is a competition between endogenous cAMP andexogenously added biotin-cAMP. The capture of cAMP is achieved by usinga specific antibody conjugated to acceptor beads.

Cell Culture and Preparation of Membranes

A stable transfected cell line and a high expressing clone were selectedfor screening. The cells were grown at 5% CO₂ in DMEM, 5% FCS, 1%Pen/Strep (Penicillin/Streptomycin) and 0.5 mg/ml of the selectionmarker G418.

Cells at approximately 80% confluence were washed 2× with PBS andharvested with Versene (aqueous solution of the tetrasodium salt ofethylenediaminetetraacetic acid), centrifuged 5 min at 1000 rpm and thesupernatant removed. The additional steps were all performed on ice. Thecell pellet was homogenised by the Ultrathurax for 20-30 s in 10 ml ofBuffer 1 (20 mM Na-HEPES, 10 mM EDTA, pH=7.4), centrifuged 15 min at20,000 rpm and the pellet resuspended in 10 ml of Buffer 2 (20 mMNa-HEPES, 0.1 mM EDTA, pH=7.4). The suspension was homogenised for 20-30s and centrifuged 15 min at 20,000 rpm. Suspension in Buffer 2,homogenisation and centrifugation was repeated once and the membraneswere resuspended in Buffer 2. The protein concentration was determinedand the membranes stored at −80° C. until use.

The assay was performed in flat-bottom 96-well plates (Costar cat. no:3693). The final volume per well was 50 μl.

Solutions and Reagents

AlphaScreen cAMP Assay Kit from Perkin Elmer Life Sciences (cat. No:6760625M); containing Anti-cAMP Acceptor beads (10 U/μl), StreptavidinDonor beads (10 U/μl) and Biotinylated-cAMP (133 U/μl).

AlphaScreen Buffer, pH=7.4: 50 mM TRIS-HCl (Sigma, cat. no: T3253); 5 mMHEPES (Sigma, cat. no: H3375); 10 mM MgCl₂, 6H₂O (Merck, cat. no: 5833);150 mM NaCl (Sigma, cat. no: S9625); 0.01% Tween (Merck, cat. no:822184). The following was added to the AlphaScreen Buffer prior to use(final concentrations indicated): BSA (Sigma, cat. no. A7906): 0.1%;IBMX (Sigma, cat. no. 15879): 0.5 mM; ATP (Sigma, cat. no. A7699): 1 mM;GTP (Sigma, cat. no. G8877): 1 uM.

cAMP standard (dilution factor in assay=5): cAMP Solution: 5.L of a 5 mMcAMP-stock+495 μL AlphaScreen Buffer.

Suitable dilution series in AlphaScreen Buffer were prepared of the cAMPstandard as well as the GLP-1 analogue or derivative to be tested, e.g.the following eight concentrations of the GLP-1 compound: 10⁻⁷, 10⁻⁸,10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ and 10⁻¹⁴M, and a series from, e.g.,10⁻⁶ to 3×10⁻¹¹ of cAMP.

Membrane/Acceptor Beads

Membranes were prepared from hGLP-1/BHK 467-12A cells with aconcentration of 6 g/well corresponding to 0.6 mg/ml (the amount ofmembranes used pr. well may vary)

“No membranes”: Acceptor Beads (15 μg/ml final) in AlphaScreen buffer

“6 μg/well membranes”: membranes+Acceptor Beads (15 μg/ml final) inAlphaScreen buffer

An aliquot (10 μl) of “No membranes” was added to the cAMP standard (perwell in duplicate wells) and the positive and negative controls

An aliquot (10 μl) of “6 μg/well membranes” was added to GLP-1 andanalogues (per well in duplicate or triplicate wells)

Pos. Control: 10 μl “no membranes”+10 μl AlphaScreen Buffer

Neg. Control: 10 μl “no membranes”+10 μl cAMP Stock Solution (50 μM)

As the beads are sensitive to direct light, any handling was in the dark(as dark as possible), or in green light. All dilutions were made onice.

Procedure

1. Make the AlphaScreen Buffer.

2. Dissolve and dilute the GLP-1/Analogues/cAMP standard in AlphaScreenBuffer.3. Make the Donor Beads Solution by mixing streptavidin donor beads (2units/well) and biotinylated cAMP (1.2 units/well) and incubate 20-30min in the dark at room temperature.4. Add the cAMP/GLP-1/Analogues to the plate: 10 μl per well.5. Prepare membrane/Acceptor Beads solution and add this to the plates:10 μl per well.6. Add the Donor Beads: 30 μl per well.7. Wrap the plate in aluminum foil and incubate on the shaker for 3hours (very slowly) at RT.8. Count on AlphaScreen—each plate pre incubates in the AlphaScreen for3 minutes before counting.

Results

The EC₅₀ [pM] values were calculated using the Graph-Pad Prism software(version 5) and are shown in Table 1 below. The potency of allderivatives in vitro was confirmed.

TABLE 1 In vitro potency Compound of Example no. EC₅₀/pM 1 26 2 43 3 624 143 5 468 6 96 7 9 8 159 9 242 10 214 11 81 12 41 13 79 14 42 15 5 1621 17 17 18 3025 19 52 20 67 21 52 22 1178 23 140 24 70 25 380 26 200 27835 28 68 29 40 30 3000 31 37 32 76

The average in vitro potency for the tested compounds (EC₅₀ average) was340 pM. Most derivatives had a good in vitro potency corresponding to anEC₅₀ of below 1200 pM.

For comparison, compound no. 13 in Table 1 of Journal of MedicinalChemistry (2000), vol. 43, no. 9, p. 1664-669 (GLP-1(7-37) acylated atK^(26,34) with bis-C12-diacid) had an in vitro potency corresponding toan EC₅₀ of 1200 pM.

Example 34: GLP-1 Receptor Binding

The purpose of this experiment is to investigate the binding to theGLP-1 receptor of the GLP-1 derivatives, and how the binding ispotentially influenced by the presence of albumin. This is done in an invitro experiment as described below.

The binding affinity of the GLP-1 derivatives of Examples 1-32 to thehuman GLP-1 receptor was measured by way of their ability to displace of¹²⁵I-GLP-1 from the receptor. In order to test the binding of thederivatives to albumin, the assay was performed with a low concentrationof albumin (0.001%—corresponding to the residual amount thereof in thetracer), as well as with a high concentration of albumin (2.0% added). Ashift in the binding affinity, IC₅₀, is an indication that the peptidein question binds to albumin, and thereby a prediction of a potentialprotracted pharmacokinetic profile of the peptide in question in animalmodels.

Conditions

Species (in vitro): Hamster

Biological End Point: Receptor Binding

Assay Method: SPA

Receptor: GLP-1 receptor

Cell Line: BHK tk-ts13

Cell Culture and Membrane Purification

A stable transfected cell line and a high expressing clone were selectedfor screening. The cells were grown at 5% CO₂ in DMEM, 10% FCS, 1%Pen/Strep (Penicillin/Streptomycin) and 1.0 mg/ml of the selectionmarker G418.

The cells (approx. 80% confluence) were washed twice in PBS andharvested with Versene (aqueous solution of the tetrasodium salt ofethylenediaminetetraacetic acid), following which they were separated bycentrifugation at 1000 rpm for 5 min. The cells/cell pellet must be kepton ice to the extent possible in the subsequent steps. The cell pelletwas homogenised with Ultrathurrax for 20-30 seconds in a suitable amountof Buffer 1 (depending on the amount of cells, but e.g. 10 ml). Thehomogenate was centrifuged at 20000 rpm for 15 minutes. The pellet wasresuspended (homogenised) in 10 ml Buffer 2 and re-centrifuged. Thisstep was repeated once more. The resulting pellet was resuspended inBuffer 2, and the protein concentration was determined. The membraneswere stored at minus 80° C.

Buffer 1: 20 mM Na-HEPES+10 mM EDTA, pH 7.4

Buffer 2: 20 mM Na-HEPES+0.1 mM EDTA, pH 7.4

Binding Assay:

SPA:

Test compounds, membranes, SPA-particles and [¹²⁵I]-GLP-1(7-36)NH₂ werediluted in assay buffer. 50 ul (micro liter) HSA (“high albumin”experiment containing 2% HSA), or buffer (“low albumin” experimentcontaining 0.001% HSA), was added to Optiplate, and 25 ul of testcompounds were added. 5-10 ug membrane protein/sample was added (50 ul)corresponding to 0.1-0.2 mg protein/ml (to be preferably optimised foreach membrane preparation). SPA-particles (Wheatgerm agglutinin SPAbeads, Perkin Elmer, # RPNQ0001) were added in an amount of 0.5 mg/well(50 ul). The incubation was started with [¹²⁵I]-GLP-1]-(7-36)NH₂ (finalconcentration 0.06 nM corresponding to 49.880 DPM, 25 ul). The plateswere sealed with PlateSealer and incubated for 120 minutes at 30° C.while shaking. The plates were centrifuged (1500 rpm, 10 min) andcounted in Topcounter.

Assay Buffer:

50 mM HEPES

5 mM EGTA

5 mM MgCl₂

0.005% Tween 20

pH 7.4

HSA was SIGMA A1653

Calculations

The IC₅₀ value was read from the curve as the concentration whichdisplaces 50% of ¹²⁵I-GLP-1 from the receptor, and the ratio of[(IC₅₀/nM) high HSA]/[(IC₅₀/nM) low HSA] was determined.

Generally, the binding to the GLP-1 receptor at low albuminconcentration should be as good as possible, corresponding to a low IC₅₀value.

The IC₅₀ value at high albumin concentration is a measure of theinfluence of albumin on the binding of the derivative to the GLP-1receptor. As is known, the GLP-1 derivatives also bind to albumin. Thisis a generally desirable effect, which extends their lifetime in plasma.Therefore, the IC₅₀ value at high albumin will generally be higher thanthe IC₅₀ value at low albumin, corresponding to a reduced binding to theGLP-1 receptor, caused by albumin binding competing with the binding tothe GLP-1 receptor.

A high ratio (IC₅₀ value (high albumin)/IC₅₀ value (low albumin)) maytherefore be taken as an indication that the derivative in questionbinds well to albumin (may have a long half-life), and also per se bindswell to the GLP-1 receptor (the IC₅₀ value (high albumin) is high, andthe IC₅₀ value (low albumin) is low).

Results

The following results were obtained, where “ratio” refers to [(IC₅₀/nM)high HSA]/[(IC₅₀/nM) low HSA]):

TABLE 2 Receptor binding affinity Compound of IC₅₀/nM IC₅₀/nM Exampleno. (low HSA) (high HSA) Ratio 1 0.19 42 219 2 0.39 320 821 3 0.29 29101 4 0.15 33 217 5 5.68 446 79 6 0.50 123 246 7 0.12 21 174 8 1.41 8057 9 0.38 60 157 10 6.49 452 70 11 0.23 213 926 12 0.16 72 453 13 0.25201 804 14 1.45 443 306 15 0.19 29 155 16 0.20 25 127 17 0.45 174 387 18373 789 2.1 19 2.38 143 60 20 0.19 333 1752 21 1.57 256 163 22 9.31 81287 23 0.85 40 47 24 2.74 50 18 25 10.9 39 3.5 26 0.38 54 143 2710.3 >1000 97 28 0.20 29 144 29 3.95 363 92 30 7.44 >1000 134 31 0.35220 629 32 0.18 369 2050

The average ratio was very good (about 300). Most derivatives had aratio above 50.

Furthermore as regards IC₅₀ (low albumin) the average IC₅₀ of thecompounds tested was 14 nM, and most derivatives were below 15.0 nM.

Finally as regards IC₅₀ (high albumin) most derivatives had an IC₅₀(high albumin) below 900 nM.

For comparison, compound no. 13 in Table 1 of Journal of MedicinalChemistry (2000), vol. 43, no. 9, p. 1664-669 (GLP-1(7-37) acylated atK^(26,34) with bis-C12-diacid) had a ratio of 51.3, an IC₅₀ (lowalbumin) of 17.7 nM, and an IC₅₀ (high albumin) of 908 nM.

Example 35: Estimate of Oral Bioavailability—Gut Injection in Rat(Caprate)

The purpose of this experiment is to estimate the oral bioavailabilityof the GLP-1 derivatives.

To this end, the exposure in plasma after direct injection into theintestinal lumen of the GLP-1 derivatives of Examples 2-17 and 19-22 wasstudied in vivo in rats, as described in the following.

The GLP-1 derivatives were tested in a concentration of 1000 uM in asolution of 55 mg/ml sodium caprate.

Male Sprague Dawley rats with a body weight upon arrival ofapproximately 240 g were obtained from Taconic (Denmark) and assigned tothe different treatments by simple randomisation, 4 rats per group. Therats were fasted for approximately 18 hours before the experiment andtaken into general anaesthesia (Hypnorm/Dormicum).

The GLP-1 derivatives were administered in the jejunum either in theproximal part (10 cm distal for the duodenum) or in the mid-intestine(50 cm proximal for the cecum). A PE50-catheter, 10 cm long was insertedinto the jejunum, forwarded at least 1.5 cm into the jejunum, andsecured before dosing by ligature around the gut and the catheter with3/0 suture distal to tip to prevent leak or catheter displacement.Catheter was placed without syringe and needle and 2 ml saline wasadministered into abdomen before closing the incision with wound clips.

100 μl of the respective GLP-1 derivative was injected into the jejunallumen through the catheter with a 1 ml syringe. Subsequently, 200 μl ofair was pushed into the jejunal lumen with another syringe to “flush”the catheter. This syringe was leaved connected to the catheter toprevent flow back into the catheter.

Blood samples (200 ul) were collected at desired intervals (usually attimes 0, 10, 30, 60, 120 and 240 min) into EDTA tubes from the tail veinand centrifuged 5 minutes, 10000 G, at 4° C. within 20 minutes. Plasma(75 ul) was separated to Micronic tubes, immediately frozen, and kept at−20° C. until analyzed for plasma concentration of the respective GLP-1derivative with LOCI (Luminescent Oxygen Channeling Immunoassay),generally as described for the determination of insulin by Poulsen andJensen in Journal of Biomolecular Screening 2007, vol. 12, p. 240-247.The donor beads were coated with streptavidin, while acceptor beads wereconjugated with a monoclonal antibody recognising a mid-/C-terminalepitope of the peptide. Another monoclonal antibody, specific for theN-terminus, was biotinylated. The three reactants were combined with theanalyte and formed a two-sited immuno-complex. Illumination of thecomplex released singlet oxygen atoms from the donor beads, which werechanneled into the acceptor beads and triggered chemiluminescence whichwas measured in an Envision plate reader. The amount of light wasproportional to the concentration of the compound.

After the blood sampling the rats were sacrificed under anaesthesia andthe abdomen was opened to verify correct catheter placement.

The mean (n=4) plasma concentrations (μmol/l) were determined as afunction of time. The ratio of plasma concentration (μmol/l) divided bythe concentration of the dosing solution (μmol/l) was calculated foreach treatment, and the results for t=30 min (30 minutes after theinjection of the compound in the jejunum) were assessed (dose-correctedexposure at 30 min) as a surrogate measure of intestinalbioavailability. The dose-corrected exposure has been shown to correlatesignificantly with the actual bioavailability.

The following results were obtained, where dose-corrected exposure at 30min refers to (the plasma concentration 30 minutes after injection ofthe compound in the jejunum (pM)), divided by (the concentration of thecompound in the dosing solution (μM)):

TABLE 3 Dose-corrected exposure at 30 min Dose-corrected Compound ofExample no. exposure at 30 min 2 98 3 67 4 39 5 124 6 93 7 99 8 86 9 6510 187 11 66 12 68 13 126 14 121 15 98 16 115 17 168 19 61 20 123 21 14022 275

All derivatives had a dose-corrected exposure at 30 min of above 38.

For comparison, compound no. 13 in Table 1 of Journal of MedicinalChemistry (2000), vol. 43, no. 9, p. 1664-669 (GLP-1(7-37) acylated atK^(26,34) with bis-C12-diacid) had a dose-corrected exposure at 30 minof 38.

Example 36: Effect on Blood Glucose and Body Weight

The purpose of the study is to verify the effect of the GLP-1derivatives on blood glucose (BG) and body weight (BW) in a diabeticsetting.

The GLP-1 derivatives are tested in a dose-response study in an obese,diabetic mouse model (db/db mice) as described in the following.

db/db mice (Taconic, Denmark), fed from birth with the diet NIH31 (NIH31M Rodent Diet, commercially available from Taconic Farms, Inc., US,see www.taconic.com), are enrolled for the study at the age of 7-9weeks. The mice are given free access to standard chow (e.g. Altromin1324, Brogaarden, Gentofte, Denmark) and tap water and kept at 24° C.After 1-2 weeks of acclimatisation, the basal blood glucose is assessedtwice on two consecutive days (i.e. at 9 am). The mice with the lowestblood glucose values are excluded from the experiments. Based on themean blood glucose values, the remaining mice are selected for furtherexperimentation and allocated to 7 groups (n=6) with matching bloodglucose levels. The mice are used in experiments with a duration of 48hours, and for up to 4 times. After the last experiment the mice areeuthanised.

The seven groups receive treatment as follows:

1: Vehicle, s.c.

2: GLP-1 derivative, 0.3 nmol/kg, s.c.3: GLP-1 derivative, 1.0 nmol/kg, s.c.4: GLP-1 derivative, 3.0 nmol/kg, s.c.5: GLP-1 derivative, 10 nmol/kg, s.c.6: GLP-1 derivative, 30 nmol/kg, s.c.7: GLP-1 derivative, 100 nmol/kg, s.c.Vehicle: 50 mM sodium phosphate, 145 mM sodium chloride, 0.05% tween 80,pH 7.4.

The GLP-1 derivative is dissolved in the vehicle, to concentrations of0.05, 0.17, 0.5, 1.7, 5.0 and 17.0 nmol/ml. Animals are dosed s.c. witha dose-volume of 6 ml/kg (i.e. 300 μl per 50 g mouse).

On the day of dosing, blood glucose is assessed at time −½ h (8.30 am),where after the mice are weighed. The GLP-1 derivative is dosed atapproximately 9 am (time 0). On the day of dosing, blood glucose isassessed at times 1, 2, 4 and 8 h (10 am, 11 am, 1 μm and 5 μm).Following the 8 h blood sampling, the mice are weighed.

On the following days, the blood glucose is assessed at time 24 and 48after dosing (i.e. at 9 am on day 2 and 3). On each day, the mice areweighed following blood glucose sampling.

The mice are weighed individually on a digital weight.

Samples for the measurement of blood glucose are obtained from the tailtip capillary of conscious mice. Blood, 10 μl, is collected intoheparinised capillaries and transferred to 500 μl glucose buffer (EKFsystem solution, Eppendorf, Germany). The glucose concentration ismeasured using the glucose oxidase method (glucose analyser Biosen 5040,EKF Diagnostic, GmbH, Barleben, Germany). The samples are kept at roomtemperature for up to 1 h until analysis. If analysis has to bepostponed, samples are kept at 4° C. for a maximum of 24 h.

ED₅₀ is the dose giving rise to half-maximal effect in nmol/kg. Thisvalue is calculated on the basis of the ability of the derivatives tolower body weight as well as the ability to lower blood glucose, asexplained below.

ED₅₀ for body weight is calculated as the dose giving rise tohalf-maximum effect on delta BW 8 hours following the subcutaneousadministration of the derivative. For example, if the maximum decreasein body weight after 8 hours is 2.0 g, then ED₅₀ bodyweight would bethat dose in nmol/kg which gives rise to a decrease in body weight after8 hours of 1.0 g. This dose (ED₅₀ body weight) may be read from thedose-response curve.

ED₅₀ for blood glucose is calculated as the dose giving rise tohalf-maximum effect on AUC delta BG 8 hours and/or 24 hours followingthe subcutaneous administration of the analogue.

The ED₅₀ value may only be calculated if a proper sigmoidaldose-response relationship exists with a clear definition of the maximumresponse. Thus, if this would not be the case the derivative in questionmay be re-tested in a different range of doses to see if a sigmoidaldose-response relationship is obtained.

Example 37: Half-Life in Minipigs

The purpose of this study is to determine the protraction in vivo of theGLP-1 derivatives after i.v. administration to minipigs, i.e. theprolongation of their time of action. This is done in a pharmacokinetic(PK) study, where the terminal half-life of the derivative in questionis determined. By terminal half-life is generally meant the period oftime it takes to halve a certain plasma concentration, measured afterthe initial distribution phase.

Male Göttingen minipigs were obtained from Ellegaard Göttingen Minipigs(Dalmose, Denmark) approximately 7-14 months of age and weighing fromapproximately 16-35 kg were used in the studies. The minipigs werehoused individually and fed restrictedly once or twice daily with SDSminipig diet (Special Diets Services, Essex, UK). After at least 2 weeksof acclimatisation two permanent central venous catheters were implantedin vena cava caudalis or cranialis in each animal. The animals wereallowed 1 week recovery after the surgery, and were then used forrepeated pharmacokinetic studies with a suitable wash-out period betweensuccessive GLP-1 derivative dosings.

The animals were fasted for approximately 18 h before dosing and from 0to 4 h after dosing, but had ad libitum access to water during the wholeperiod.

The GLP-1 derivatives were dissolved in 50 mM sodium phosphate, 145 mMsodium chloride, 0.05% tween 80, pH 7.4 to a concentration of usuallyfrom 20-60 nmol/ml. Intravenous injections (the volume corresponding tousually 1-2 nmol/kg, for example 0.033 ml/kg) of the compounds weregiven through one catheter, and blood was sampled at predefined timepoints for up till 13 days post dosing (preferably through the othercatheter). Blood samples (for example 0.8 ml) were collected in EDTAbuffer (8 mM) and then centrifuged at 4° C. and 1942 G for 10 minutes.Plasma was pippetted into Micronic tubes on dry ice, and kept at −20° C.until analyzed for plasma concentration of the respective GLP-1 compoundusing ELISA or a similar antibody based assay or LC-MS. Individualplasma concentration-time profiles were analyzed by a non-compartmentalmodel in Phoenix WinNonLin ver. 6.2. (Pharsight Inc., Mountain View,Calif., USA), and the resulting terminal half-lives (harmonic mean)determined.

Results

The derivative of Example 2 was tested and it had a half-life of 87hours.

For comparison, compound no. 13 in Table 1 of Journal of MedicinalChemistry (2000), vol. 43, no. 9, p. 1664-669 (GLP-1(7-37) acylated atK^(26,34) with bis-C12-diacid) had a half-life of 5 hours.

Example 38: Effect on Food Intake

The purpose of this experiment was to investigate the effect of GLP-1derivatives on food intake in pigs. This was done in a pharmacodynamic(PD) study as described below, in which food intake was measured from 1to 4 days after administration of a single dose of the GLP-1 derivative,as compared to a vehicle-treated control group.

Female Landrace Yorkshire Duroc (LYD) pigs, approximately 3 months ofage, weighing approximately 30-35 kg were used (n=3-4 per group). Theanimals were housed in a group for approximately 1 week duringacclimatisation to the animal facilities. During the experimental periodthe animals were placed in individual pens at least 2 days before dosingand during the entire experiment for measurement of individual foodintake. The animals were fed ad libitum with pig fodder (SvinefoderDanish Top) at all times both during the acclimatisation and theexperimental period. Food intake was monitored on line by logging theweight of fodder every 15 minutes. The system used was Mpigwin(Ellegaard Systems, Faaborg, Denmark).

The GLP-1 derivatives are dissolved in a phosphate buffer (50 mM sodiumphosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4) atconcentrations of 12, 40, 120, 400 or 1200 nmol/ml corresponding todoses of 0.3, 1, 3, 10 or 30 nmol/kg. The phosphate buffer serves asvehicle. Animals are dosed with a single subcutaneous dose of the GLP-1derivative or vehicle (dose volume 0.025 ml/kg) on the morning of day 1,and food intake is measured for 1-4 days after dosing. On the last dayof each study, 1-4 days after dosing, a blood sample for measurement ofplasma exposure of the GLP-1 derivative is taken from the heart inanaesthetised animals. The animals are thereafter euthanised with anintra-cardial overdose of pentobarbitone. Plasma content of the GLP-1derivatives is analysed using ELISA or a similar antibody based assay,or LC-MS.

Food intake is calculated as mean±SEM 24 h food intake on each of theexperimental days. Statistical comparisons of the 24 hour food intake inthe vehicle vs. GLP-1 derivative group are done using two-way-ANOVArepeated measures, followed by Bonferroni post-test.

The compound of Example 10 was tested in a dose of 3 nmol/kg, and inthis dose no effect was seen on food intake. The compound of Example 2was tested in a dose of 3 nmol/kg and showed a significant reduction offood intake on both days of the experiment (23% on day 1, and 35% on day2).

Example 39: Pharmacokinetics in rat

The purpose of this Example is to investigate half-life in vivo in rat.

In vivo pharmacokinetic studies in rats were performed with the GLP-1derivatives of Examples 2, 10, 17-18, and 31, as described in thefollowing. Semaglutide was included for comparison.

Male Sprague Dawley rats of same age with a body weight of approximately400 g were obtained from Taconic (Denmark) and assigned to thetreatments by simple randomisation on body weight, approximately 4 ratsper group, so that all animals in each group were of similar bodyweight.

The GLP-1 derivatives (approximately 6 nmol/ml) were dissolved in 50 mMsodium phosphate, 145 mM sodium chloride, 0.05% tween 80, pH 7.4.Intravenous injections (1.0 ml/kg) of the compounds were given through acatheter implanted in the right jugular vein. Blood was sampled fromvena sublingualis for 5 days post dosing. Blood samples (200 μl) werecollected in EDTA buffer (8 mM) and then centrifuged at 4° C. and 10000G for 5 minutes. Plasma samples were kept at −20° C. until analyzed forplasma concentration of the respective GLP-1 compound.

The plasma concentrations of the GLP-1 compounds were determined using aLuminescence Oxygen Channeling Immunoasssay (LOCI), generally asdescribed for the determination of insulin by Poulsen and Jensen inJournal of Biomolecular Screening 2007, vol. 12, p. 240-247. The donorbeads were coated with streptavidin, while acceptor beads wereconjugated with a monoclonal antibody recognising a mid-/C-terminalepitope of the peptide. Another monoclonal antibody, specific for theN-terminus, was biotinylated. The three reactants were combined with theanalyte and formed a two-sited immuno-complex. Illumination of thecomplex released singlet oxygen atoms from the donor beads, which werechanneled into the acceptor beads and triggered chemiluminescence whichwas measured in an Envision plate reader. The amount of light wasproportional to the concentration of the compound.

Plasma concentration-time profiles were analyzed using Phoenix WinNonLinver. 6.2, Pharsight Inc., Mountain View, Calif., USA), and the half-life(T ) calculated using individual plasma concentration-time profiles fromeach animal.

Results

The half-life of semaglutide tested in the same set-up (but with n=8)was 11 hours.

TABLE 4 Half-life in rat Compound of Example no. t½/h 2 26 10 23 17 1018 10 31 19

The tested derivatives of the invention had a half-life that was similaror better to that of semaglutide.

Example 40: Estimate of Oral Bioavailability—Gut Injection and OralGavage in Rat (SNAC)

The purpose of this experiment is to estimate the oral bioavailabilityof the GLP-1 derivatives in a rat model. In brief, a liquid solution ofthe GLP-1 derivative in sodium N-[8-(2-hydroxybenzoyl)amino]caprylate(SNAC) is administered by gut injection (to the intestines), or by oralgavage (to the stomach), and the subsequent exposure in plasma of theGLP-1 derivative is measured.

A 250 mg/ml stock solution of SNAC is prepared by dissolving SNAC (12.5g) in highly pure laboratory water (MilliQ) (50.0 ml). The pH isadjusted to about 8.5 with 1 N NaOH (aq).

Solutions with about 1000 uM (800-1200 uM) of the GLP-1 derivatives in250 mg/ml SNAC are prepared by dissolving the desired amount of therespective GLP-1 derivative in the SNAC stock solution. Theconcentration of the GLP-1 derivative is determined prior toadministration by a state-of-the-art method, such as CLND-HPLC(chemiluminescent nitrogen detection for HPLC).

32 male Sprague Dawley rats with a body weight upon arrival ofapproximately 240 g are obtained from Taconic (Denmark) and assigned tothe different treatments by simple randomisation, 8 rats per group. Allrats are fasted on grids for approximately 18 hours before theexperiment.

For gut injection, on the day of experiment, rats are taken into generalanaesthesia (Hypnorm/Dormicum) and remained anaesthetized during theentire experiment. The GLP-1 derivatives of Examples 5-7 areadministered in the proximal part of the jejunum (10 cm distal for theduodenum). A PE50-catheter, 10 cm long, is inserted into the jejunum,forwarded at least 1.5 cm into the jejunum, and secured before dosing byligature around the gut. Furthermore, the catheter is provided with a3/0 suture distal to tip to prevent leak or catheter displacement. Thecatheter is placed without syringe and needle and 2 ml saline isadministered into abdomen before closing the incision with wound clips.

100 μl SNAC solution of the respective GLP-1 derivative is injected intothe jejunal lumen through the catheter with a 1 ml syringe.Subsequently, 200 μl of air is pushed into the jejunal lumen withanother syringe to “flush” the catheter. This syringe is left connectedto the catheter to prevent flow back into the catheter.

Blood samples (200 ul) are collected at desired intervals (usually attimes 0, 30, 60, 120 and 180 min) into EDTA tubes from the tail vein.

For oral gavage, the animals are conscious during the entire experiment.

100 μl SNAC solution of the GLP-1 derivatives is administered by oralgavage directly to the stomach.

Blood samples (200 ul) are collected at desired intervals (usually attimes 0, 30, 60, 120 and 180 min) into EDTA tubes from the sublingualplexus.

All obtained blood samples are kept on ice and centrifuged for 5minutes, 10000 G, at 4° C. within 20 minutes. Plasma (75 ul) isseparated to Micronic tubes, immediately frozen, and kept at −20° C.until analyzed for plasma concentration of the respective GLP-1derivative with LOCI (Luminescent Oxygen Channeling Immunoassay),generally as described for the determination of insulin by Poulsen andJensen in Journal of Biomolecular Screening 2007, vol. 12, p. 240-247.The donor beads are coated with streptavidin, while acceptor beads areconjugated with a monoclonal antibody recognising a mid-/C-terminalepitope of the peptide. Another monoclonal antibody, specific for theN-terminus, is biotinylated. The three reactants are combined with theanalyte and formed a two-sited immuno-complex. Illumination of thecomplex released singlet oxygen atoms from the donor beads, which arechanneled into the acceptor beads and triggered chemiluminescence whichis measured in an Envision plate reader. The amount of light isproportional to the concentration of the compound.

After the blood sampling all rats are sacrificed under anaesthesia andthe abdomen of the gut injection rats is opened to verify correctcatheter placement.

The mean (n=8) plasma concentrations (μmol/l) are determined as afunction of time. The AUC of the plasma exposure (μmol/l) vs time curve,from time 30 to 180 (min), is dose-corrected, i.e., divided by theamount (dose) of the derivative in the dosed solution (pmol). The thusdose-corrected AUC of plasma exposure from time 30-180 min (having theunit of min×pM/pmol=min/L) is used as a surrogate measure ofbioavailability—a measure to rank the derivatives with regards to theirabsorption in the rat model.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

1. A derivative of a GLP-1 analogue, which analogue comprises a first Kresidue at a position corresponding to position 27 of GLP-1(7-37) (SEQID NO: 1); a second K residue at a position corresponding to position Tof GLP-1(7-37), where T is an integer in the range of 7-37 except 18 and27; and a maximum of ten amino acid changes as compared to GLP-1(7-37);wherein the first K residue is designated K²⁷, and the second K residueis designated K^(T); which derivative comprises two protracting moietiesattached to K²⁷ and K^(T), respectively, via a linker, wherein theprotracting moiety is selected from Chem. 2 and Chem. 1:HOOC—C₆H₄—O—(CH₂)_(y)—CO—*  Chem. 2:HOOC—(CH₂)_(x)—CO—*,  Chem. 1: in which x is an integer in the range of6-16, and y is an integer in the range of 3-17; and the linker comprisesChem. 5:

wherein k is an integer in the range of 1-5, and n is an integer in therange of 1-5; or a pharmaceutically acceptable salt, amide, or esterthereof.
 2. The derivative of claim 1, wherein the linker furthercomprises a Glu di-radical selected from Chem. 6, and/or Chem. 7:


3. The derivative of claim 1, wherein the linker is attached to theepsilon-amino group of the first or the second K residue.
 4. Thederivative of claim 1, wherein T is 12, 20, 22, 24, 36, or
 37. 5. Thederivative of claim 1, wherein the analogue comprises no K residuesother than the first and the second K residue.
 6. The derivative ofclaim 1, wherein x is
 12. 7. The derivative of claim 1, wherein y is 9or
 11. 8. The derivative of claim 1, wherein k is
 1. 9. The derivativeof claim 1, wherein n is
 1. 10. The derivative of claim 1, wherein theanalogue comprises a GLP-1 analogue of Formula I:Xaa₇-Xaa₈-Glu-Gly-Thr-Xaa₁₂-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Xaa₂₄-Xaa₂₅-Xaa₂₆-Lys-Phe-Ile-Xaa₃₀-Xaa₃₁-Leu-Val-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈s-Xaa₃₉,  FormulaI: wherein Xaa₇ is L-histidine, imidazopropionyl, α-hydroxy-histidine,D-histidine, desamino-histidine, 2-amino-histidine,P3-hydroxy-histidine, homohistidine, N^(α)-acetyl-histidine,N^(α)-formyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine,3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine; Xaa₈ is Ala,Gly, Val, Leu, Ile, Thr, Ser, Lys, Aib, (1-aminocyclopropyl) carboxylicacid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl) carboxylic acid,(1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylicacid; Xaa₁₂ is Lys or Phe; Xaa₁₆ is Val or Leu; Xaa₁₈ is Ser, Arg, Asn,Gln, or Glu; Xaa₁₉ is Tyr or Gln; Xaa₂₀ is Leu, Lys, or Met; Xaa₂₂ isGly, Glu, Lys, or Aib; Xaa₂₃ is Gln, Glu, or Arg; Xaa₂₄ is Ala or Lys;Xaa₂₅ is Ala or Val; Xaa₂₆ is Val, His, or Arg; Xaa₃₀ is Ala, Glu, orArg; Xaa₃₁ is Trp or His; Xaa₃₄ is Glu, Asn, Gly, Gln, or Arg; Xaa₃₅ isGly, Aib, or absent; Xaa₃₆ is Arg, Gly, Lys, or absent; Xaa₃₇ is Gly,Ala, Glu, Pro, Lys, Arg, or absent; Xaa₃₈ is Ser, Gly, Ala, Glu, Gln,Pro, Arg, or absent; and Xaa₃₉ is Gly or absent.
 11. A compoundaccording to claim 1, selected from the following: Chem. 50, Chem. 51,Chem. 52, Chem. 53, Chem. 54, Chem. 55, Chem. 56, Chem. 57, Chem. 58,Chem. 59, Chem. 60, Chem. 61, Chem. 62, Chem. 63, Chem. 64, Chem. 65,Chem. 66, Chem. 67, Chem. 68, Chem. 69, Chem. 70, Chem. 71, Chem. 72,Chem. 73, Chem. 74, Chem. 75, Chem. 76, Chem. 77, Chem. 78, Chem. 79,Chem. 80, and Chem. 81; or a pharmaceutically acceptable salt, amide, orester thereof.
 12. A compound according to claim 1, characterised by itsname, and selected from a listing of each of the names of the compoundsof Examples 1-32 herein, or a pharmaceutically acceptable salt, amide,or ester thereof.
 13. A derivative according to claim 1, for use as amedicament.
 14. A derivative according to claim 1, for use in thetreatment and/or prevention of all forms of diabetes and relateddiseases, such as eating disorders, cardiovascular diseases,gastrointestinal diseases, diabetic complications, critical illness,and/or polycystic ovary syndrome; and/or for improving lipid parameters,improving β-cell function, and/or for delaying or preventing diabeticdisease progression.
 15. A method for treating or preventing all formsof diabetes and related diseases, such as eating disorders,cardiovascular diseases, gastrointestinal diseases, diabeticcomplications, critical illness, and/or polycystic ovary syndrome;and/or for improving lipid parameters, improving β-cell function, and/orfor delaying or preventing diabetic disease progression—by administeringa pharmaceutically active amount of a derivative according to claim 1.